ov 


'State  Hygiene 


PRACTICAL  BACTERIOLOGY,  MICROBIOLOGY 
AND  SERUM  THERAPY 

(MEDICAL   AND   VETERINARY) 


PRACTICAL 

BACTERIOLOGY,  MICROBIOLOGY 
AND  SERUM  THERAPY 

(MEDICAL  AND   VETERINARY) 
A    TEXT  BOOK  FOR  LABORATORY   USE 


BY 


DR  A. 


FORMERLY   DIRECTOR   OF   BACTERIOLOGICAL    LABORATORIES   OF   THE    MILITARY 

HOSPITALS    OF    FRANCE    AND    OF    THE    P^AN    HOSPITAL. 

LAUREATE   OF   THE    FRKNCH    INSTITUTE 


TRANSLATED 

AND   ADAPTED   FROM   THE   FIFTH   FRENCH   EDITION 
BY 

H.  J.  HUTCHENS,  D.S.O. 

M.A.,  M.R.C.S.,  L.R.C.r.,   D.  P.  H.  (OXFORD) 

HEATH    PROFESSOR   OF  COMPARATIVE    PATHOLOGY   AND   BACTERIOLOGY    OF   THE 

UNIVERSITY   OF   DURHAM  ;    FORMERLY    AN    ASSISTANT   SCIENTIFIC 

INVESTIGATOR,    ROYAL   COMMISSION   ON   TUBERCULOSIS 


WITH  416   ILLUSTRATIONS,   149   OF   WHICH  ARE  COLOURED 


LONGMANS,    GREEN,    AND    CO. 

39   PATERNOSTER   ROW,   LONDON 

NEW  YORK,    BOMBAY,    AND   CALCUTTA 

1913 

All  rights  reserved 


BIOLOGY  IIBR. 


5+ 


BIOLOGY 
LIBRARY 


PKEFACE  TO  THE  ENGLISH  EDITION. 

So  far  as  the  aim  and  scope  of  the  book  are  concerned  they  are  sufficiently 
described  in  the  Author's  prefaces.  It  remains  to  be  said  that  the  lack  of 
a  similar  text  book  in  English,  at  once  sufficiently  simple  to  put  into  the 
hands  of  the  beginner  and  at  the  same  time  sufficiently  advanced  to  be  of 
service  to  the  more  experienced  observer,  together  with  the  evident  popularity 
of  Dr.  Besson's  work  in  French  speaking  countries,  seems  to  be  sufficient 
justification  for  preparing  the  present  translation. 

A  mere  translation  however  of  a  book  dealing  with  so  rapidly  advancing 
a  science  as  Microbiology  would  have  been  hardly  satisfactory,  hence  an 
attempt  has  been  made  to  bring  it  up  to  date  by  incorporating  matter  which 
has  appeared  since  the  French  edition  went  to  press. 

As  regards  the  translation  itself  the  original  text  has  been  followed  as  closely 
as  possible,  but  the  aim  throughout  has  been  to  reproduce  the  sense  rather 
than  the  actual  words. 

The  alterations  which  have  been  made  may  be  included  under  two  heads, 
namely  :  alterations  in  the  text  and  alterations  in  the  arrangement  of  the 
text.  With  two  exceptions  the  whole  of  the  French  text  has  been  followed. 
In  Chap.  VII.  the  theory  of  the  Microscope  has  been  rewritten  as,  generally 
speaking,  the  Student  seems  to  possess  but  a  very  limited  knowledge  of  the 
instrument  and  it  was  thought  that  an  amplification  of  the  French  text 
would  be  useful.  I  wish  to  thank  Mr.  A.  S.  Percival,  Senior  Surgeon  to  the 
Eye  Infirmary,  Newcastle  upon  Tyne,  for  the  help  he  has  given  me  in  this 
part  of  the  subject.  Chaps.  XXV.,  XXVL,  and  XXVII.  dealing  with  the 
Paratyphoid  bacilli  have  also  been  rewritten  in  view  of  the  work  of  the  Royal 
Army  Medical  Corps  in  India  and  of  Dr.  F.  A.  Bainbridge  in  England. 

There  are  many  notes  and  additions.  Thus  for  instance  it  was  found 
necessary  to  incorporate  the  important  results  obtained  by  the  Royal  Com- 
mission on  Tuberculosis.  All  such  notes  and  additions  are  clearly  indicated 
either  by  a  footnote  or  by  being  enclosed  withia  square  brackets.  It  should 
be  said  also  that  in  a  few  cases  where  the  authorities  were  in  favour  of  a 
different  nomenclature  from  that  until  recently  in  use  the  new  names  have 
been  substituted  thus  Discomyces  appears  as  the  generic  name  in  place  of 
Streptothrix. 


vi  PREFACE  TO  THE   ENGLISH   EDITION 

The  arrangement  of  the  text  in  the  translation  varies  from  that  of  the 
original  in  some  respects.  In  the  first  place  the  subject  matter  has  been 
divided  into  seven  Parts  instead  of  three.  Secondly,  the  arrangement  of 
the  Parts  has  been  subjected  to  some  modification.  To  take  the  Bacteria 
for  example  it  seemed  that  in  a  book  intended  for  use  in  the  laboratory  it 
might  be  an  advantage  if  these  organisms  were  arranged  morphologically 
and  then  subdivided  according  to  their  staining  reactions  and  cultural  charac- 
teristics. The  plan  adopted  can  be  readily  seen  by  a  reference  to  the  Table 
of  Contents.  Of  course  no  classification  is  perfect  and  therefore  free  from 
criticism  but  after  a  good  deal  of  consideration  it  was  felt  that  practical 
usefulness  merited  the  attempt. 

Reference  to  any  particular  point  will  present,  I  hope,  no  difficulty.  In 
addition  to  an  Index  and  a  very  full  Table  of  Contents,  a  summary  of  the 
subject  matter  heads  the  various  Chapters.  These  of  course  are  quite  inde- 
pendent of  the  French  edition. 

The  illustrations  have  been  carefully  revised.  Many  of  them  are  new 
though  illustrating  familiar  subjects  and  were  drawn  by  my  former  laboratory 
attendant,  Mr.  H.  Boot,  under  my  supervision  from  preparations  in  my 
laboratory.  Some  were  drawn  by  Mr.  Richard  Muir.  For  others  I  am 
indebted  to  the  courtesy  of  the  Controller  of  His  Majesty's  Stationery  Office, 
of  Professor  G.  H.  F.  Nuttall,  F.R.S.,  of  Dr.  H.  G.  Adamson,  and  of  the 
Publishers  of  Mense's  Handbuch  der  Tropenkrankheiten.  Miss  M.  V.  Lebour, 
M.Sc.  of  the  Zoological  Department  of  the  University  of  Leeds  kindly 
undertook  to  redraw  the  whole  of  the  line  drawings. 

In  the  preparation  of  this  translation  I  wish  to  acknowledge  my  very  par- 
ticular indebtedness  to  Professor  G.  A.  Lebour,  M.A.,  D.Sc.  who  has  given 
me  at  all  times  most  invaluable  assistance.  To  my  former  colleague  Mr. 
C.  F.  Fox  who  had  charge  of  the  records  of  the  Royal  Commission  on  Tubercu- 
losis I  owe  many  thanks  for  the  considerable  care  with  which  he  undertook 
the  thankless  task  of  reading  over  the  whole  of  the  MS.  before  it  went  to 
press  and  for  revising  the  proofs. 

H.  J.  HUTCHENS. 

Newcastle  upon  Tyne, 

March  3lst,  1913. 


PEEFACE  TO  THE  FIFTH  FRENCH  EDITION. 

EECENT  advances  in  Microbiology  have  necessitated  an  entire  revision  of 
the  text.  While  still  retaining  its  original  form  most  of  the  chapters  have 
been  recast  and  much  new  matter  has  been  incorporated. 

The  plan  adopted  when  the  book  was  first  written  of  omitting  all  discussion 
upon  matters  of  theory  has  been  adhered  to  but  it  has  nevertheless  been 
thought  desirable  to  include  a  Chapter  on  Immunity  and  the  Properties 
of  Immune  Serums.  The  object  of  this  has  been  to  explain  as  clearly  and 
simply  as  possible  the  principles  underlying  the  phenomena  of  agglutination, 
of  the  fixation  of  the  complement  and  the  opsonic  index  and  to  describe  the 
practical  details  in  such  a  manner  as  to  enable  the  Student  to  become  familiar 
with  the  technique  employed  in  these  delicate  investigations  and  so  be  in  a 
position  to  appreciate  the  more  detailed  monographs. 

In  view  of  its  importance  in  clinical  diagnosis  a  description  of  the  Ultra- 
microscope  has  been  included. 

Numerous  additions  and  alterations  have  been  made  in  the  second  Part. 
Most  of  the  chapters  have  been  supplemented.  The  serum  treatment  of 
Dysentery  and  of  meningococcal  Meningitis  has  been  described  as  fully  as 
was  consistent  with  the  scope  of  the  work.  The  anaerobic  micro-organisms, 
the  paratyphoid  bacilli,  Sporotrichosis,  Syphilis,  etc.  are  all  subjects  of 
additions  while  many  modifications  have  been  introduced  into  the  descrip- 
tion and  classification  of  the  parasitic  Protozoa,  especially  the  Piroplasmata, 
Leishmania  and  Trypanosomala. 

As  in  former  editions  the  sole  object  has  been  to  write  a  clear  and  concise 
account  of  each  subject  and  one  which  will  be  abreast  of  recent  knowledge 
retaining  at  the  same  time  those  characteristic  features  of  the  book  which 
have  been  the  subject  of  favourable  comment  both  here  and  abroad. 

A.  BESSON. 
15^  May,  1911. 


PEEFACE  TO  THE  FIRST  FRENCPI  EDITION. 

So  important  a  place  does  Microbiology  now  occupy  in  the  medical  curriculum 
that  not  only  are  laboratories  fully  equipped  for  research  and  teaching  to  be 
found  in  all  medical  Schools,  but  the  Student  on  leaving  his  School  should 
have  at  least  sufficient  knowledge  of  the  subject  to  carry  out  for  himself 
the  more  simple  investigations,  such,  for  instance,  as  the  recognition  of  the 
tubercle  bacillus  and  the  detection  of  the  diphtheria  bacillus. 

The  present  work  has  been  designed  purely  as  a  laboratory  guide,  the  one 
object  constantly  in  view  in  its  preparation  having  been  to  make  it  a  true 
vade  mecum — a  book  which  would  both  direct  the  beginner  step  by  step  and, 
at  the  same  time,  afford  to  the  more  skilled  worker  such  assistance  as  would 
enable  him  to  pursue  his  researches  in  a  profitable  direction. 

My  experience  as  a  Teacher  of  Microbiology  and  as  a  Director  of  labora- 
tories has  I  venture  to  think  given  me  the  qualifications  necessary  for  the 
task  in  hand. 

All  matters  of  theory  and  all  references  to  original  sources  have  been 
studiously  avoided  since  adequate  information  upon  these  matters  is  forth- 
coming in  the  many  excellent  Text  books  of  Bacteriology. 

In  the  first  Part  of  the  book  the  methods  applicable  to  micro-organisms  in 
general  are  detailed  and  while  in  each  chapter  a  number  of  methods,  all  of 
which  have  been  recommended  by  various  authorities,  are  described,  em- 
phasis is  laid  upon  those  with  which  I  have  obtained  the  most  satisfactory 
results  and  which  I  feel  may  confidently  be  recommended  to  the  beginner. 

The  second  Part  is  concerned  with  a  description  of  the  methods  most 
suitable  to  the  various  different  micro-organisms.  The  Bacteria  are  described 
first  and  then  the  parasitic  Fungi  and  Protozoa  the  importance  of  which, 
however  considerable  it  now  may  be,  threatens  to  occupy  an  even  greater 
place  in  the  Pathology  of  the  future. 

The  third  Part  which  completes  the  book  is  devoted  to  a  short  account 
of  the  methods  available  for  the  bacteriological  examination  of  water  and  air. 

Much  care  has  been  bestowed  upon  the  illustrations,  and  in  order  that  the 
figures  may  be  of  as  much  use  as  possible  to  the  Student  in  interpreting  his 
own  results  they  were  drawn  and  coloured  by  myself  from  my  own  pre- 
parations and  faithfully  represent  the  appearances  which  should  be  obtained 
if  the  directions  in  the  text  are  carefully  followed. 


x  PREFACE  TO   THE   FIRST   FRENCH   EDITION 

I  wish  to  take  this  opportunity  of  expressing  my  thanks  to  those  of  my 
Teachers  to  whom  I  am  indebted  for  my  instruction  in  the  subject ;  I 
have  drawn  largely  upon  them  and  should  this  book  be  received  with  some 
favour  I  shall  be  not  unmindful  of  those  to  whom  the  credit  is  due. 

A.  BESSON. 

15th  October,  1897. 


CONTENTS. 
PART   I.     GENERAL   TECHNIQUE. 

CHAP.  PAGE 

I.  Sterilization. 

Introduction,  -  3 

Section  I.     Sterilization  by  dry  heat,  4 

(1)  Sterilization  in  a  naked  flame,  p.  4.  (2)  Sterilization  by  hot  air, 
p.  4. 

Section  II.     Sterilization  by  moist  heat,  -  7 

(1)  Sterilization  in  steam  at  100°  C.,  p.  7.  (2)  Sterilization  in 
steam  under  pressure,  p.  9.  (3)  Sterilization  by  discontinuous 
heating,  p.  12. 

Section  III.     Sterilization  by  filtration,  -  14 

(1)  Filtration  of  water,  p.  15.  (2)  Filtration  of  culture  media, 
p.  18.  (3)  The  filtration  of  small  quantities  of  liquid,  p.  24. 

Section  IV.     Sterilization  by  antiseptics,  26 

II.  Culture  media. 

Introduction,  -  28 

Section  I.     Liquid  media,  30 

(1)  Media  made  from  animal  tissues  and  fluids,  p.  30.  (2)  Media 
made  from  vegetable  tissues,  p.  37.  (3)  Synthetic  media,  p.  38. 

Section  II.     Solid  media,       -  39 

(1)  Gelatin  media,  p.  39.  (2)  Agar  media,  p.  42.  (3)  Media  made 
from  albuminous  fluids  and  tissues,  p.  45.  (4)  Vegetable  media, 
p.  55.  (5)  Coloured  media,  p.  56. 

III.  Incubators. 

Introduction,  -  58 

Section  I.     Devices  for  automatically  regulating  the  temperature 

of  incubators,  -  59 

Section  II.     Incubators  heated  by  coal  gas,  61 

Section  III.     Incubators  heated  by  electricity,  65 

Section  IV.     Incubators  heated  by  petrol,  gasoline,   or  petro- 
leum oil,  66 


xii  CONTENTS 

IV.  The     methods    of    sowing    and    cultivating    aerobic 

organisms. 

Introduction,  -  67 

Section  I.     Instruments  used  for  sowing  cultures,  -  67 

Section  II.     The  methods  of  solving  cultures,  70 

Section  III.     Conditions  essential  to  satisfactory  growth,  72 

Section  IV.     The  examination  of  cultures,  73 

Section  V.     The  methods  of  storing  cultures,  -  75 

V.  The    isolation    of    aerobic    micro-organisms    in    pure 

culture. 

Introduction,  -  -        76 

Section  I.     Mechanical  methods,     -  76 

(1)  Isolation  by  dilution,  p.  76.  (2)  Isolation  by  dissemination, 
p.  77. 

Section  II.     Biological  methods,  83 

(1)  Heat,  p.  84.  (2)  Cultivation  at  the  optimum  temperature, 
p.  84.  (3)  Cultivation  on  special  media,  p.  85.  (4)  Animal  inocu- 
lation, p.  85. 

VI.  The    cultivation    and    isolation    of    anaerobic    micro- 

organisms. 

Introduction,  -  87 

Section    I.     The    methods    of    abstracting    air    from    culture 

media,     -  87 

(1)  By  boiling,  p.  87.  (2)  By  displacing  the  air  with  an  inert 
gas,  p.  88.  (3)  By  absorbing  the  oxygen,  p.  89.  (4)  By  the  use  of 
a  vacuum,  p.  90.  (5)  Tests  for  oxygen,  p.  92. 

Section  II.     The  cultivation  of  anaerobic  organisms,       -  92 

(1)  In  liquid  media,  p.  92.     (2)  In  solid  media,  p.  99. 

Section  III.     The  isolation  of  anaerobic  organisms,  -       101 

(1)  Plate  method,  p.  101.     (2)  Tube  method,  p.  103. 

Section  IV.     Vacuum  incubators,  -  104 

VII.  The  Microscope. 

Introduction,  -  106 

Section  I.     The  microscope  stand,  -  106 

Section  II.     The  optical  parts  of  the  microscope,  -  -       107 

A.  The  objectives,  p.  107.  (1)  Magnification,  p.  107.  (2)  Spherical 
aberration,  p.  110.  (3)  Angular  aperture,  p.  112.  (4)  Numerical 
aperture,  p.  112.  (5)  Resolving  power,  p.  112.  (6)  Brightness  of 
image,  p.  113.  (7)  Definition,  p.  114.  (8)  Chromatic  aberration, 
p.  114.  (9)  Flatness  of  image,  p.  115.  B.  The  eyepieces,  p.  116. 

Section  III.     The  care  of  the  microscope,       -  -       117 

Section  IV.     The  method  of  using  the  microscope,  -       118 


CONTENTS  xiii 

Section  V.     The  measurement  of  microscopical  objects,    -  121 

(1)  The  experimental  determination  of  the  magnification  produced  by 
a  system  of  lenses,  p.  121.  (2)  The  measurement  of  objects  under  the 
microscope,  p.  122. 

Section  VI.     Dark-ground  illumination,  -  123 

(1)  The  application  of  dark-ground  illumination  to  micro-biology, 
p.  124.  (2)  The  construction  of  the  dark-ground  illuminator,  p.  124. 
(3)  The  method  of  using  the  instrument,  p.  125. 

VIII.  The  microscopical  examination  of  cultures  of  micro- 

organisms. 

Introduction,  -  130 

Section  I.     The  preparation  of  slides  and  cover -glasses,  -  130 

Section  II.     The  examination  of  unstained  preparations,  131 

(1)  The  examination  of  a  culture  on  an  ordinary  slide,  p.  132.  (2) 
Hanging-drop  preparations,  p.  132. 

Section  III.     The  examination  of  stained  preparations,  -         -       135 

(1)  Staining  solutions,  p.  137.  (2)  Simple  staining,  p.  140.  (3)  Gram's 
stain,  p.  142.  (4)  Claudius'  method,  p.  144. 

IX.  The    staining    of  spores,  capsules   and  flagella.    The 

study  of  the  motility  of  bacteria. 

Section  I.     Spores,  -      145 

(1)  The  examination  of  unstained  preparations,  p.  145.  (2)  The 
staining  of  spores,  p.  146. 

Section  II.     The  staining  of  capsules,  -       147 

Section  III.     The  staining  of  flagella,  148 

(1)  The  staining  of  flagella  in  living  organisms,  p.  148.  (2)  The 
staining  of  flagella  in  dried  preparations,  p.  149. 

Section    IV.      The     methods     of    studying     the     motility    of 

micro-organisms,      -  -       154 

X.  Animal  inoculation. 

Section  I.     The  selection  of  animals  for  inoculation,  156 

Section  II.     The  keeping  of  animals,     -  157 

Section    III.      The     spontaneous     diseases     of    experimental 

animals,  159 

Section  IV.     The  handling  of  experimental  animals,  160 

Section  V.     Experimental  inoculations,  -  -       165 

(1)  Instruments,  p.  165.  (2)  Preparation  of  the  material  for  inocula- 
tion, p.  169.  (3)  Technique  of  inoculation,  p.  170. 

Section  VI.     Observations  to  be  made  on  inoculated  animals,        182 


XI.  Post  mortem  examinations. 

Introduction,  - 

(1)  Instruments,  p.  184.  (2)  Preliminary  operations,  p.  185.  (3) 
Examination  of  the  external  surface  of  the  carcase,  p.  185.  (4)  Exami- 
nation of  the  internal  organs,  p.  185.  (5)  Removal  of  tissues  for 
histological  examination,  p.  188. 


XIV 


CONTENTS 


XII.  The  collection  of  material  for  bacteriological  exami- 

nation, -  -      191 

(1)  Hair,  p.  191.  (2)  Skin,  p.  191.  (3)  Sputum,  p.  191.  (4)  Blood, 
p.  192;  The  collection  of  serum,  p.  195.  (5)  Pharyngeal  exudates, 
p.  197.  (6)  Abscesses,  p.  197.  (7)  Aqueous  humour,  p.  197.  (8) 
Pleural  and  pulmonary  exudates,  p.  198.  (9)  Ascitic  fluid,  p.  198. 
(10)  Tumours  and  lymphatic  glands,  p.  198.  (11)  Splenic  puncture, 
p.  198  ;  Splenectomy,  p.  199.  (12)  Lumbar  puncture,  p.  199.  (13) 
Milk,  p.  201.  (14)  Urine,  p.  201.  (15)  Stools,  p.  202. 

XIII.  The  bacteriological  examination  of  fluids  and  tissues. 

Section  I.     Film  preparations,  -       203 

(1)  Unstained  preparations,  p.  203.  (2)  Stained  preparations:  A. 
Preparation  of  films,  p.  204.  B.  Staining  methods — (i)  Simple 
staining,  p.  205.  (ii)  Differential  staining,  p.  207. 

Section  II.     Histological  preparations,    -  211 

(1)  Instruments,  p.  211.  (2)  Freezing  methods,  p.  212.  (3)  Paraffin 
embedding  methods,  p.  212.  (4)  Preliminary  treatment  of  sections, 
p.  215.  (5)  The  staining  of  sections  :  A.  Simple  staining,  p.  216. 
B.  Differential  staining,  p.  217. 

XIV.  Immunity.    The  properties  of  immune  serums. 

Introduction,  -  -       221 

The  mechanism  of  immunity,  p.  222. 

Section  I.     Prophylactic  and  therapeutic  serums,  223 

Section  II.     Antitoxins,  224 

Section  III.     Agglutinins,  -       225 

The  mechanism  of  agglutination,  p.  226. 

Section  IV.     Bactericidal  properties,       -  227 

The  mechanism  of  bacteriolysis,  p.  228  ;  Hsemolysins,  p.  230 ;  The 
preparation  of  an  hsemolytic  serum,  p.  230 ;  The  mechanism  of 
haemolysis,  p.  231 ;  The  fixation  of  the  complement,  p.  232 ;  The 
technique  of  the  complement-fixation  reaction,  p.  233. 

Section  V.     Opsonins,    -  -       239 

The  method  of  determining  the  opsonic  index,  p.  240. 


PART  II.    THE  PATHOGENIC  BACTERIA. 

SUB- DIVISION  I.     THE   NON-SPORE-BEARING,  GRAM-POSITIVE, 
NON-ACID-FAST  BACILLI. 

XV.  Bacillus  diphtherias. 

Introduction,  -  -  _       245 

Section  I.     Experimental  inoculation,     -         -  -       247 

(1)  The  symptoms  and  .lesions  produced  in  animals  susceptible  to 
infection,  p.  247.  (2)  The  influence  of  other  organisms  on  the  clinical 
course  of  the  disease,  p.  249. 

Section  II.     Morphology,  250 


CONTENTS  xv 

Section  III.     Biological  properties,  254 

(1)  Vitality  and  virulence,  p.  254.  (2)  Bio-chemical  reactions,  p.  256. 
(3)  Toxin — Preparation,  p.  257  ;  Testing  and  storing,  p.  260  ;  Action 
on  animals,  p.  260 ;  Nature  and  properties,  p.  261.  (4)  Vaccination, 
p.  262.  (5)  Serum  therapy— Antitoxin,  its  preparation,  p.  265 ; 
Properties,  p.  266  ;  Standardization,  p.  267 ;  Serum  therapeutics, 
p.  268.  (6)  Agglutination,  p.  269. 

Section    IV.     The    detection,    isolation    and    identification    of 

the  diphtheria  bacillus,     -  -      269 

(1)  The  collection  of  material,  p.  270.  (2)  Methods  of  examination, 
p.  270.  (3)  Summary  of  diagnostic  tests,  p.  273. 

Bacillus  pseudo-diphtheria  (Hofmann's  bacillus),  273 

XVI.  Bacillus  pyocyaneus. 

Introduction,  -  -      276 

Section  I.     Experimental  inoculation,      -  -       276 

Section  II.     Morphology,  277 

Section  III.     Biological  properties,  279 

Section  IV.     Detection  and  isolation  of  the  organism,  281 

XVII.  The  bacillus  of  swine  erysipelas. 

Introduction,  -  -       283 

Section  I.  Experimental  inoculation,  284 

Section  II.     Morphology,  284 

Section  III.     Biological  properties,  286 

Section    IV.     The    detection,    isolation    and    identification    of 

the  bacillus,     -  -      287 

The  bacillus  of  mouse  septicaemia,  p.  288. 


SUB-DIVISION    II.     THE  NON-SPORE-BEARING,  GRAM-POSITIVE, 
ACID-FAST   BACILLI. 

XVIII.  Bacillus  tuberculosis. 

Introduction,  -  -       289 

(1)  Types  of  tubercle  bacilli,  p.  2S9.  (2)  Human  tuberculosis, 
p.  292.  (3)  Tuberculosis  in  the  lower  animals,  p.  294.  (4)  Asso- 
ciated micro-organisms,  p.  297. 

Section  I.     Experimental  inoculation,     -  297 

A.  Guinea-pigs,  p.  297.  B.  Rabbits,  p.  300.  C.  Dogs,  p.  302. 
D.  Cattle,  p.  303.  E.  Birds,  p.  304.  F.  Cold-blooded  vertebrata, 
p.  305. 

Section  II.     Morphology, 

(1)  Microscopical  appearance,  p.  305;  Staining  methods,  p.  306. 
A.  The  staining  of  films,  p.  307.  B.  The  staining  of  sections, 
p.  310 ;  The  appearance  of  stained  bacilli,  p.  312.  (2)  Cultural 
characteristics,  p.  314. 

b 


xvi  CONTENTS 

Section  III.     Biological  properties,  322 

(1)  Viability  and  virulence,  p.  322.  (2)  Toxins,  p.  323.  A.  Toxic 
properties  of  dead  bacilli,  p.  323.  B.  Koch's  old  tuberculin,  p.  324. 
C.  Tuberculins  T.A.,  T.O.,  T.R.,  p.  328.  D.  Maragliano's  tuber- 
culin,' p.  329.  E.  Toxalbumin,  p.  329.  (3)  Vaccination,  p.  330. 
(4)  Serum  therapy,  p.  334.  (5)  Agglutination,  p.  335.  (6)  Immune 
body,  p.  337. 

Section  IV.     The  detection  of  the  tubercle  bacillus,  -       337 

A.  Sputum,  p.  339.  B.  Blood,  p.  341.  G.  Pus,  p.  342.  D. 
Exudates,  p.  342.  E.  Granulomata,  p.  343.  F.  Nasal  cavities, 
p.  343.  0.  Urine,  p.  343.  H.  Excreta,  p.  344.  /.  Milk,  p.  345. 

The  paratubercle  or  acid-fast  bacilli,  345 

XIX.  Bacillus  leprse. 

Introduction,  -  -  348 

Section  I.     Attempts  to  reproduce  the  disease  experimentally,  -  348 

Section  II.     Morphology,  350 

Section  III.     Serum  therapy,  -  -  353 

Section  IV.     Detection  and  identification  of  the  leprosy  bacillus,  354 


SUB-DIVISION   III.     THE   NON-SPORE-BEARING,  GRAM-NEGATIVE 
BACILLI  THAT   DO  NOT   LIQUEFY  GELATIN. 

XX.  Bacillus  dysenteriae  epidemicae. 

Introduction,  -  -  356 

Section  I.     Experimental  inoculation,  -                                    -  357 

Section  II.     Morphology,  *  358 

Section  III.     Biological  properties,  -       359 

(1)  Biochemical  reactions,  p.  359.  (2)  Vitality,  p.  360.  (3)  Toxin, 
p.  361.  (4)  Vaccination  and  serum  therapy,  p.  361.  (5)  Agglutina- 
tion, p.  363.  (6)  Precipitins,  p.  363.  (7)  Immune  body,  p.  363. 

Section  IV.     Detection,    isolation    and    identification    of    the 

dysentery  bacillus,    -  -       364 

Serum  diagnosis  of  dysentery,  p.  364. 

The  bacillus  dysentericus,  El.  Tor  No.  1,  365 

XXI.  Bacillus  febris  entericse. 

Introduction,  -  366 

Section  I.     Experimental  inoculation,     -  -       367 

A.  Inoculation  of  viruses  of  ordinary  virulence,  p.  368.  B.  Inocula- 
tion of  viruses  of  exalted  virulence,  p.  368.  C.  Infection  by  the 
alimentary  canal,  p.  369. 

Section  II.     Morphology,  -       370 

Section  III.     Biological  properties,  -  373 

(1)  Biochemical  reactions,  p.  373.  (2)  Variability  of  flagella,  p.  376. 
(3)  Viability  and  virulence,  p.  376.  (4)  Toxins,  p.  376.  (5)  Vaccina- 


CONTENTS  xvii 

tion,  p.  380.  (6)  Serum  therapy,  p.  383.  (7)  Agglutination — 
Serum-diagnosis  of  enteric  fever,  p.  384.  Application  of  agglutina- 
tion reaction  to  the  identification  of  the  typhoid  bacillus,  p.  389. 
(8)  Absorption  of  agglutinins,  p.  389.  (9)  Complement  fixation, 
p.  390. 

Section    IV.     Detection,    isolation    and    identification    of    the 

typhoid  bacillus,       -  -      390 

XXII.  Bacillus  coli. 

Introduction,  -  -      393 

Section  I.     Experimental  inoculation,  -       394 

Section  II.     Morphology,  395 

Section  III.     Biological  properties,  396 

(1)  Biochemical  reactions,  p.  396.  (2)  Variability  of  flagella,  p.  398. 
(3)  Vitality  and  virulence,  p.  398.  (4)  Toxin,  p.  398.  (5)  Vaccina- 
tion and  serum  therapy,  p.  399.  (6)  Agglutination,  p.  399. 

Section  IV.     Detection,  isolation  and  identification  of  the  colon 

bacillus,  -  -      400 

The  bacillus  of  green  diarrhoea,  400 

4 
« 

XXIII.  The  isolation  of  the  typhoid  and  colon  bacilli  from  water, 

stools,  etc.,  and  the   methods    of  identifying   the   two 
organisms. 

Introduction,  -  -      401 

Section  I.     The  isolation  of  the  typhoid  and  colon  bacilli,  402 

(1)  Original  methods,  p.  402.  (2)  Eisner's  method  and  its  modifica- 
tions, p.  403.  (3)  Precipitation  methods,  p.  406.  (4)  Methods  based 
upon  the  motility  of  the  typhoid  bacillus,  p.  406.  (5)  Chantemesse's 
carbolic  media,  p.  407.  (6)  Conradi-Drigalski's  method,  p.  407.  (7) 
Endo's  medium,  p.  408.  (8)  Caffeine  media,  p.  408.  (9)  Malachite 
green  media,  p.  409.  (10)  China  green  medium,  p.  410.  (11)  Bile 
media,  p.  410.  (12)  Brilliant  green  medium,  p.  411.  (13)  Neutral 
red  media,  p.  411.  (14)  Methods  based  upon  agglutination,  p.  412. 
MacConkey's  media,  p.  412. 

Section  II.     The  identification  of  the  typhoid  and  colon  bacilli,  -      412 

XXIV.  The  pneumobacillus  of  Friedlander. 

Introduction,  -  415 

Section  I.     Experimental  inoculation,  -      416 

Section  II.     Morphology,  416 

Section  III.     Biological  properties,  417 

Section    IV.     Detection,    isolation    and    identification    of    the 
pneumobacillus,  - 

The  bacillus  of  rhmoscleroma, 
The  bacillus  of  ozaena,  - 


xviii  CONTENTS 

XXV.  The  paratyphoid  bacilli. 

The  origin  of  the  terms  "paratyphoid"  and  "paracolon." — The 
relation  of  the  "paratyphoid"  bacilli  to  the  "  haemorrhagic  septic- 
aemia" group  and  to  the  "  enteritidis "  group. — The  classification 
adopted. — The  origin  and  definition  of  the  "Salmonella  group." — 
Other  names  suggested  for  the  "paratyphoid  "  group,  -  420 

XXVI.  Bacillus  paratyphosus  A. 

Introduction,  -  -       423 

Section  I.     Experimental  inoculation.,  -                                     -       424 

Section  II.     Morphology,  424 

Section  III.     Biological  properties,  -       424 

(1)  Biochemical  reactions,  p.  424.  (2)  Vitality  and  virulence,  p.  425. 
(3)  Toxin,  p.  425.  (4)  Vaccination,  p.  425.  (5)  Agglutination, 
p.  426.  (6)  Absorption  tests,  p.  427.  (7)  Complement  fixation, 
p.  428. 

Section  IV.     The  diagnosis  of  paratyphoid  A  infections.     The 

isolation  and  identification  of  the  bacillus,  428 

XXVII.  The  Salmonella  group. 

1.  Bacillus  paratyphosus  B. 

Introduction,  -  431 

Section  I.     Experimental  inoculation,     -  433 

Section  II.     Morphology,  433 

Section  III.     Biological  properties,          -  -       434 

(1)  Biochemical  reactions,  p.  434.  (2)  Toxins,  p.  434.  (3)  Vaccina- 
tion and  the  properties  of  immune  serums,  p.  435.  (4)  Agglutination, 
p.  435.  (5)  Absorption  tests,  p.  436.  (6)  Complement  fixation, 
p.  437. 

Section  IV.     The  isolation  and  identification  of  the  bacillus,  -       437 

2.  Bacillus  enteritidis  Aertrycke. 

Introduction,  -  -  438 

Section  I.     Experimental  infection,  -  439 

Section  II.     Morphology,  -  440 

Section  III.     Biological  properties,  440 

Section  IV.     Isolation  and  identification  of  the  bacillus,          -  441 

3.  Bacillus  enteritidis  Gaertner. 

Introduction,  -  -      442 

Section  I.     Experimental  inoculation,     -  442 

Section  II.     Morphology,  443 

Section  III.     Biological  properties,  443 

Section  IV.     Isolation  and  identification  of  the  bacillus,          -       444 

4.  Pseudo-Gaertner  bacilli,    -  ....  .       444 


CONTENTS  xix 

5.  Bacillus  typhi  murium,  .      444 

6.  Danysz's  virus,  .       444 

7.  The  bacillus  of  psittacosis,  .       445 

8.  Bacillus  icteroides,    -  .      445 

XXVIII.  The  pasteurella  group  of  bacilli. 

Introduction,  -                                              -  445 

I.  Pasteurella  gallinae,  .      447 
Section  I.     Experimental  inoculation,  -      448 
Section  II.     Morphology,                                             -  449 
Section  III.     Biological  properties,  -       451 

Section  IV.     The  isolation  and  identification  of  the  bacillus,  -      452 
Similar  organisms  in  epizootics  among  other  birds,  p.  452. 

II.  Pasteurella  cuniculi,  .      453 

III.  Pasteurella  suis,  -      454 

IV.  Pasteurella  bovis,    -  -      455 
V.  Pasteurella  ovis,  -      456 

VI.  Pasteurella  caprae,  -      456 

VII.  Pasteurella  equi,  456 

VIII.  Pasteurella  canis,  -      457 

M'Gowan's  bacillus  of  distemper,  p.  459. 

IX.  Immunization  with  polyvalent  vaccines,  459 

XXIX.  Bacillus  pestis. 

Introduction,  -  -      460 

Section  I.     The  experimental  disease,     -  -      463 

Section  II.     Morphology,  -      464 

Section  III.     Biological  properties,  466 

(1)  Vitality  and  virulence,  p.  466.     (2)  Biochemical  reactions,  p.  467. 

(3)   Toxin,    p.    467.     (4)  Vaccination,    p.    468.     (5)  Serum  therapy, 
p.  471.     (6)  Agglutination,  p.  472.     (7)  Precipitins,  p.  473. 

Section     IV.     Isolation    and    identification    of    the  plague 

bacillus,  -  -      473 

Post  mortem  appearances  in  naturally  infected  rats,  p.  474. 

XXX.  Micrococcus  melitensis. 

Introduction,  -  -      475 

Section  I.     Experimental  inoculation,     -  -      476 

Section  II.     Morphology,  -      476 

Section  III.     Biological  properties,  -      477 

Section    IV.     Detection,    isolation    and    identification  of   the 

organism,  -      478 


xx  CONTENTS 

XXXI.  Bacillus  mallei. 

Introduction,  -  -      480 

Section  I.     Experimental  inoculation,  -      480 

Section  II.     Morphology,  -       482 

Section  III.     Biological  properties,  484 

Section  IV.     Detection  and  isolation  of  the  bacillus,  486 
The  diagnosis  of  glanders,  p.  486. 

SUB-DIVISION  IV.     NON-SPORE-BEARING,  GRAM- NEGATIVE  BACILLI 
THAT   LIQUEFY   GELATIN. 

XXXII.  Vibrio  cholerae  asiaticse. 

Introduction,  -      488 

Section  I.     The  experimental  disease,  489 

Section  II.     Morphology,  491 

Section  III.     Biological  properties,  -      493 

(1)  Vitality  and  virulence,  p.  493.     (2)  Biochemical  reactions,  p.  494. 

(3)    Toxin,   p.   494.     (4)    Vaccination,   p.   496.     (5)    Serum  therapy, 

p.   498.      (6)    Bactericidal     properties — Agglutination,    p.  499.     (7) 
Complement  fixation,  p.  500. 

Section    IV.     Detection,    isolation    and    identification  of   the 

vibrio,       -  -      500 

The  vibrio  of  Finkler  Prior,  -  502 

The  vibrio  of  Deneke,    -  503 

Vibrio  metchnikowi,  503 

SUB-DIVISION  V.     NON-SPORE-BEARING,   GRAM-NEGATIVE  BACILLI 
THAT  DO  NOT  GROW  ON  GELATIN. 

XXXIII.  Pfeiffer's  influenza  bacillus. 

Introduction,  -  -       504 

Section  I.     Experimental  inoculation,  505 

Section  II.     Morphology,  506 

Section  III.     Biological  properties,  508 

Section  IV.     Detection  and  isolation  of  the  organism,  510 

The  hsemoglobinophilic  bacilli.  510 

The  bacillus  of  whooping  cough  (Bordet-Gengou).  -      511 

XXXIV.  The  bacillus  of  soft  sore. 

Introduction,  -  -       513 

Section  I.     Experimental  infection,  -       513 

Section  II.     Morphology,  514 

Section  III.     Biological  properties,  -       515 

Section  I V.     Detection,  isolation  and  identification  of  the  bacillus,       515 


CONTENTS  xxi 


SUB-DIVISION  VI.     THE   SPORE-BEARING,   GRAM-POSITIVE, 
AEROBIC  BACILLI. 

XXXV.  Bacillus  anthracis. 

Introduction,  -  -       517 

Section  I.     The  experimental  disease,  518 

(1)  Susceptible  and  immune  animals,  p.  518.  (2)  Methods  of  inocula- 
tion, p.  519.  (3)  Symptoms  and  lesions  in  experimental  animals, 
p.  519. 

Section  II.     Morphology,  -      520 

(1)  Microscopical  appearance  and  staining  reactions,  p.  520.  (2) 
Cultural  characteristics,  p.  524. 

Section  III.     Biological  properties,  -      525 

(1)  Viability  and  resistance,  p.  525.  (2)  Virulence,  p.  527.  (3) 
Toxin,  p.  529.  (4)  Serum  therapy,  p.  530.  (5)  Agglutination, 
p.  533. 

Section  I V.     Detection,  isolation  and  identification  of  the  anthrax 

bacillus,  -  -      533 

Examination  of  carcases  dead  of  anthrax,  p.  534.  Isolation  of  the 
bacillus  from  soil,  535. 


SUB-DIVISION  VII.     THE   SPORE-BEARING,  GRAM-POSITIVE, 
ANAEROBIC  BACILLI. 

XXXVI.  Bacillus  tetani. 

Introduction,  -  -      536 

Section  I.     Experimental  inoculation,  -       536 

(1)  Inoculation  of  soil  or  pus,  p.  537.     (2)  Inoculation  of  pure  cul- 
tures, p.  537.     (3)  Inoculation  of  spores,  p.  538. 

Section  II.     Morphology,  539 

Section  III.     Biological  properties,  -      541 

(1)  Vitality  and  virulence,  p.  541.     (2)  Toxin,  p.  541.     (3)  Vaccina- 
tion, p.  544.     (4)  Serum  therapy,  p.  545.     (5)  Agglutination,  p.  548. 

Section  IV.     Detection,  isolation  and  identification  of  the  tetanus 

bacillus,  -  -      548 

Bacillus  botulinus,  p.  549. 

XXXVII.  The  bacillus  of  quarter  ill. 

Introduction,  -  •-.-.-  552 

Section  I.     The  experimental  disease,  -                                    -  552 

Section  11.     Morphology,  ~  554 

Section  III.     Biological  properties,  -       556 

(1)  Vitality   and   virulence,    p.    556.     (2)  Vaccination,    p.    556.     (3) 
Toxin,  p.  558.     (4)  Serum  therapy,  p.  559.     (5)  Agglutination,  p.  560. 


xxii  CONTENTS 

XXXVIII.  Bacillus  maligni  cedematis. 

Introduction,  -  -       561 

Section  I.     The  experimental  disease,  -       561 

Section  II.     Morphology,  563 

Section  III.     Biological  properties,  -       565 

SUB-DIVISION   VIII. 

XXXIX.  Certain  anaerobic  micro-organisms  found  in  gan- 
grenous suppurations. 

I.  Bacillus  perfringens,  569 

The  bacillus  of  Ghon  and  Sachs,  p.  571. 

II.  Bacillus  pseudo-oedema,  571 

III.  Bacillus  ramosus,  -  571 

IV.  Bacillus  serpens,  572 
V.  Bacillus  thetoides,  572 

VI.  Bacillus  fragilis,  573 

VII.  Bacillus  fusiformis,  574 

VIII.  Spirillum  riigrum,  -  577 

IX.  Staphylococcus  parvulus,  578 

X.  Micrococcus  fetidus,  578 

XI.  Bacillus  aerobicus  sepis,  578 

SUB-DIVISION  IX.     THE  GRAM-POSITIVE  MICROCOCCI. 

XL.  The  pneumococcus. 

Introduction,  -  -       580 

Section  I.     Experimental  inoculation,  581 

Section  II.     Morphology,  582 

Section  III.     Biological  properties,  -       585 

(1)  Vitality  and  virulence,  p.  585.  (2)  Biochemical  reactions,  p.  586. 
(3)  Toxins,  p.  586.  (4)  Vaccination,  p.  587.  (5)  Serum  therapy, 
p.  588.  (6)  Agglutination,  p.  589.  (7)  Precipitins,  p.  590.  (8) 
Immune  bodies,  p.  590. 

Section    IV.     Detection,    isolation    and    identification    of    the 

pneumococcus,  -      590 

XLI.  Streptococci  hominis. 

Introduction,  -         -  592 

Varieties  of  streptococci,  p.  593. 

Section  I.     Experimental  inoculation,  594 

Section  II.     Morphology,  -       595 


CONTENTS  xxiii 

Section  III.     Biological  properties,  .      599 

(1)  Vitality  and  virulence  p.  599.  (2)  Biochemical  reactions,  p.  600  ; 
Andrewes  and  Herder's  classification,  p.  601.  (3)  Toxins,  p.  602; 
Streptocolysin,  p.  603.  (4)  Vaccination,  p.  604.  (5)  Serum  therapy, 
p.  605  ;  Monovalent  serums,  p.  606  ;  Polyvalent  serums,  p.  608.  (6) 
Agglutination,  p.  609.  (7)  Bordet-Gengou  reaction,  p.  609. 

Section  IV.     Detection  and  isolation  of  streptococci,  -       609 

The  streptococcus  of  Bonome,  .       610 

XLII.  Streptococci  animalium. 

I.  The  streptococcus  of  strangles,  .  61 1 

II.  The  streptococcus  of  contagious  mammitis  of  cows,  -  613 

III.  The  micrococcus  of  contagious  mammitis  of  ewes,1  -  615 

XLIII.  Staphylococci  pyogenetes. 

Introduction,  -  -       617 

Section  I.     Experimental  inoculation,  .       618 

Section  II.  Morphology,  .       619 

Section  III.     Biological  properties,  .       620 

(1)  Viability  and  virulence,  p.  620.  (2)  Bio-chemical  reactions, 
p.  621.  (3)  Toxin,  p.  622.  (4)  Vaccination,  p.  623.-  (5)  Serum 
therapy,  p.  624.  (6)  Agglutination,  p.  624. 

Section    IV.     Detection,    isolation    and    identification    of   the 

staphylococci.  .        _       625 

The  diplococcus  crassus,  _       626 

XLIV.  The  enterococcus. 

Introduction,  -  -  627 

Section  I.     Experimental  inoculation,  -  627 

Section  II.     Morphology  and  biological  properties,  -  628 

(1)  Microscopical  appearance,  p.  628.  (2)  Cultural  characteristics, 
p.  628.  (3)  Vitality  and  virulence,  p.  629. 

Section  III.     The  detection,  isolation  and  identification  of  the 

enterococcus,  -       629 

XLV.  Micrococcus  tetragenus. 

Introduction,  -  631 

Section  I.     Experimental  inoculation,  -                                     -       631 

Section  II.     Morphology,  632 

Section  III.     Biological  properties,  632 

Section    IV.     Detection,    isolation    and    identification    of   the 

organism,  -       633 

1  Though  morphologically  not  belonging  to  the  group  it  will  be  convenient  to  include 
the  micrococcus  in  the  same  chapter  as  the  streptococcus  causing  mammitis  in  cows 


xxiv  CONTENTS 

SUB-DIVISION  X.     THE  GRAM-NEGATIVE   MICROCOCCI. 

XLVI.  The  gonococcus. 

Introduction,  -  -       634 

Section  I.     Experimental  inoculation,  -       635 

Section  II.     Morphology,  635 

(1)    Microscopical   appearance   and   staining   reactions,    p.    635.     (2) 
Cultural  characteristics,  p.  638. 

Section  III.     Biological  properties,  -.      640 

Section  IV.     Detection  and  isolation  of  the  gonococcus,  -  642 

XL VII.  The  meningococcus. 

Introduction,  ••  644 

Relationship  of  the  meningococcus  to  the  gonococcus. 

Section  I.     Experimental  inoculation,  645 

Section  II.     Morphology,  645 

Section  III.     Biological  properties,  647 

Section  IV.     The  isolation  and  identification  of  the  meningo- 
coccus,    -  -       650 

Micrococcus  catarrhalis.       -  -      651 


PART   III.     THE   PARASITIC   FUNGI. 

XL VIII.  The  parasitic  Hypomycetes. 

Section  I.     The  genus  Discomyces,  -  655 

Introduction. 

I.  Discomyces  bovis,     -  656 

(1)  Experimental  inoculation,   p.   656.     (2)  Morphology;    Detection 
of  the  parasite.     A.   Microscopical  appearance,  p.  657.     B.  Cultural 
characteristics,  p.  659.     (3)  Biological  properties,  p.  660. 
The  parasites  of  actinomycosis,  p.  660. 

II.  Discomyces  israeli,  p.  661.     III.  Discomyces  thibiergi,  p.  661. 

IV.  Discomyces  liquefaciens,  p.  661.  V.  Discomyces  garteni, 
p.  661.  VI.  Discomyces  asteroides,  p.  662.  VII.  Discomyces 
forsteri,  p.  662.  VIII.  Discomyces  rosenbachi,  p.  662.  IX. 
Discomyces  madurse,  662.  X.  Discomyces  freeri,  p.  664. 
XI.  Discomyces  brasiliensis,  p.  665. 
The  parasites  of  mycetoma,  p.  665. 

XII.  Discomyces  minutissimus,  p.  666.  XIII.  Discomyces  far- 
cinicus,  p.  667.  XIV.  Discomyces  caprae,  p.  668.  XV.  Dis- 
comyces hofmanni,  p.  669.  XVI.  The  polychrome  discomyces 
of  Vall<§e,  p.  669. 


CONTENTS 


XXV 


Section  II.     The  genus  Malassezia,  -  669 

Section  III.     The  genus  Trichosporum,  -  -  670 

Section  IV.     The  genus  Coccidioides,  -         -  671 

Section  V.     The  genus  Sporotrichum,  -  672 

Section  VI.     The  genus  Oidium,  -  674 

Section  VII.     Of  unknown  classification.  -       674 

The  parasite  of  Bursattee. 

XLIX.  The  parasitic  Phycomycetes.     Parasites  of  the  family 
Mucoracidae. 

Introduction,  -  -  575 

General  methods  of  examination,  cultivation,  etc. 

Section  I.     The  genus  Mucor,  .  676 

Section  II.     The  genus  Lichtheimia,  -         -  677 

Section  III.     The  genus  Rhizomucor,  -  678 

Section  IV.     The  genus  Rhizopus,  -  $7$ 

L.    The   parasitic   Ascomycetes.     Parasites    of  the    family 
Gymnoascidse. 

Section  I.     The  genus  Trichophyton,      -  -      679 

General  methods  of  examination,  cultivation,  etc. 

A.  Endothrix  species,  -      682 

(1)  Trichophyton  tonsurans,  p.  682.  (2)  Trichophyton  sabouraudi, 
p.  684.  (3)  Trichophyton  violaceum,  p.  685.  (4)  Trichophyton 
sulphureum,  p.  685. 

B.  Endo-ectothrix  species,  -      685 

(1)    Trichophyton     mentagrophytes,      p.      685.       (2)    Trichophyton 
equinum,  p.   687.     (3)  Trichophyton   caninum,  p.    687.     (4)  Tricho- 
phyton felineum,    p.   687.     (5)   Trichophyton  megnini,  p.   687.     (6)    . 
Trichophyton    favitorme,    p.    687.     (7)  Trichophyton   concentricum, 

p.  687. 

Section  II.     The  genus  epidermophyton,  688 

Epidermophyton  cruris,  p.  688. 

Section  III.     The  genus  Microsporum,  -  -      688 

(1)  Microsporum  audouini,  p.  688. 

Section  IV.     The  genus  Achorion,  -       690 

A.  The  human  parasite — Achorion  schcenleini,  690 

B.  The  parasites  of  favus  in  the  lower  animals,  -  -       692 

Section  V.     The  genus  Lophophyton,  692 

Lophophyton  gallinse,  p.  692. 

Section  VI.     Micro-organisms  in  Alopecia  areata,  -      692 

Section  VII.     The  bacillus  of  seborrhoza  oleosa,      -  -       692 


xxvi  CONTENTS 

LI.  The  parasitic  Ascomycetes  (continued).     Parasites  of  the 
family  Perisporacidae. 

Introduction,  -  -      694 

General  methods  of  examination,  cultivation,  etc. 

Section  I.     The  genus  Aspergillus,  695 

(1)  Aspergillus  glaucus,  p.  695.  (2)  Aspergillus  repens,  p.  695.  (3) 
Aspergillus  malignus,  p.  695.  (4)  Aspergillus  fumigatus,  p.  695. 
(5)  Aspergillus  pictor,  p.  698. 

Section  II.     The  genus  Sterygmatocystis,  -       699 

Section  III.     The  genus  Penicillum,  -       700 

Section  IV.     The  parasite  of  Tinea  imbricata,  -       700 

LII.  The  parasitic  Ascomycetes  (continued).    Parasites  of  the 
family  Saccharomycetidae. 

Introduction,  -  -       701 

Section  I.     The  genus  Endomyces, — Endomyces  albicans,  .  701 

Section  II.     The  genus  Saccharomyces ,  -  •    -       704 

(1)  Saccharomyces  tumefaciens,  p.  704.  (2)  Other  species  of  Saccharo- 
myces, p.  705. 

Section  III.     The  genus  Cryptococcus,   -  -      706 

Section  IV.     The  Saccharomyces  and  Cancer,  -       707 


PART  IV.     THE   PATHOGENIC  SPIROCHiET^. 

LIII.  The  blood-inhabiting  spirochsetes. 

A.  Human  spirochsetosis. 

Introduction,  -  711 

Section  I.     Experimental  inoculation,     -  713 

Section  II.     Morphology  and  methods  of  detection,  714 

(1)    Microscopical   appearance   and   staining   reactions,    p.    714.     (2) 
Cultivation  of  the  parasites,  p.  715. 

Section  III.     Serum  therapy,  -      716 

The  differentiation  of  the  various  human  spirochtetes,  p.  717- 

B.  Spirochaetosis  in  the  lower  animals,  -  717 

(1)  Spirochseta  anserina,  p.  717.     (2)  Spirochseta  marchouxi,  p.  718. 
(3)  Spirochseta  theileri,  p.  719. 

LIV.  The  treponema  pallidum. 

Introduction,  -  720 

Section  I.     Experimental  inoculation,  -       721 

Experiments  on  immunization,  p.  7'24. 
Section  II.     Morphology,  ••       725 

(1)  Microscopical  appearance,  p.  725.     (2)  Staining  methods,  p.  726. 
a.  Films,  p.  727.     /3.  Flagellum  staining,  p.  730.     7.  Sections,  p.  730. 


CONTENTS  xxvii 

Section  III.     Detection  and  identification  of  the  treponema,    •  732 

Section  IV.     Cultivation  experiments,  736 

Section  V.     Serum  diagnosis,                                                      -  737 

Wassermann's  reaction,  p.  737.      Chemical  methods  of  serum  diag- 
nosis, p.  740. 


PART  V.     THE  PROTOZOAN  PARASITES. 

I.  THE    RHIZOPODA. 

LV.  The  amoebae. 

Introduction,  -  745 

Section  I.     Amoeba  princeps,  745 

Section  II.     The  intestinal  amoebce,  746 

(i)  Amoeba  coli,  p.  747.     (ii)  Amoeba  histolytica,  p.  748. 

Methods  of  detection,  p.   748.     Cultivation,  p.    750.     Experimental 
infection,  p.  751. 

II.  THE   SPOROZOA. 
A.     THE  NEOSPORIDIA. 

LVI.  The  Microsporidia,  Myxosporidia,  Sarcosporidia,  Hap- 
losporidia. 

Section  I.     The  Microsporidia,  752 

Nosema  bombycis,  752.     Nosema  apis,  753. 

Section  II.     The  Myxosporidia,  754 

Section  III.     The  Sarcosporidia,  -       756 

Section  IV.     The  Haplosporidia,    -  759 

B.     THE   TELOSPORID1A. 

LVII.  The  Coccidiidea. 

Section  I.     The  genus  Coccidium,  -  -      760 

Coccidium    cuniculi,    p.    760.     Morphology,   p.    761.     Life    history, 

p.  762. 

Other  principal  species  of  coccidia,  p.  764. 

Section  II.     The  genus  Klossia,     -  765 

Section  III.     Parasites  in  tumours,  -      766 

(1)  Coccidia  p.  766.     (2)  Micrococcus  neoformans,  p.  769. 

LVIII.  The  intra-corpuscular  Haematozoa. 

Section  I.     The  genus  hcemamoeba,  -      770 


xxviii  CONTENTS 

1.  The  haematozoon  of  malaria,   -  -       770 

Methods  of  examination,  p.  770.  Structure  of  the  parasite,  p.  772. 
Morphology,  p.  774.  Life  history,  p.  776.  The  different  species  of 
haematozoa  found  in  malaria,  p.  778. 

Examination  of  mosquitos,  p.  780. 
Experimental  inoculation,  p.  781. 

2.  The  haematozoon  of  monkeys,    -  781 

3.  The  hsematozoon  of  bats,  -  781 

4.  The  haematozoa  of  birds,  -  781 
Section  II.     The  genus  Hcemogregarina,  -       783 

1.  Haemogregarina  stepanowi,  783 

2.  Haemogregarina  ranarum,  -  784 

3.  Haemogregarina  lacertarum,  785 

LIX.  The  intra-corpuscular  haematozoa  (continued}. 

Section  III.     The  genus  Piroplasma,     -  786 

1.  Piroplasma  bigeminum,   -  787 

Morphology  and  method  of  multiplication,  p.  787.  Methods  of 
examination,  p.  790.  Immunity,  p.  791. 

2.  Piroplasma  ovis,      -  791 

3.  Piroplasma  canis,  791 

4.  Piroplasma  equi,  792 

5.  Piroplasma  pitheci,  793 
Section  IV.     The  genus  Theileria,  793 

Theileria  parva. 

LX.  The  Gregarinida.  794 

C.     OF   UNCERTAIN    CLASSIFICATION. 

LXI.  Parasites  of  the  genus  Leishmania. 

1.  Leishmania  donovani,  797 

Methods  of  detection  and  appearance  of  the  parasite  in  the  tissues, 
p.  797.  Appearance  in  cultures,  p.  799.  ^Etiology,  p.  799.  Experi- 
mental inoculation,  p.  800. 

2.  Leishmania  infantum,  800 

3.  Leishmania  tropica,  -  -  -        -  -      802 


III.     THE  FLAGELLATA. 

LXII.  The  Flagellata. 

Introduction,  -  -       803 

Section  I.     The  Trypanosomata*     -  803 

Introduction  and  general  methods  of  examination,  p.  803. 


CONTENTS  xxix 

1.  Trypanosoma  lewisi.     The  rat  trypanosome,  -       805 

Trypanosomes  in  rodents  other  than  rats,  p,  808. 

2.  Trypanosoma  equiperdum.     The  trypanosome  of  Dourine,        809 

3.  Trypanosoma  brucei.     The  trypanosome  of  Nagana,-  811 

African  trypanosomiases  related  to  nagana,  p.  813. 

4.  Trypanosoma  evansi.     The  trypanosome  of  Surra,  814 

5.  Trypanosoma    equinum.     The    trypanosome    of    Mai    de 

Caderas,  814 

6.  Trypanosoma  theileri.     The  trypanosome  of  Galziekte,  816 

7.  The  trypanosomes  of  Sleeping  Sickness,       -  -       816 

Trypanosoma  gambiense,  p.  816. 
Trypanosoma  rhodesiense,  p.  820. 

8.  Trypanosoma  cruzi,   -  822 

9.  Trypanosomes  in  birds,  823 
10.  Trypanosomes  in  cold-blooded  vertebrata,  824 

Section  II.     Trichomonas  vaginalis,  825 
Other  species  of  Trichomonas,  p.  826. 

Section  III.     Lamblia  intestinalis,                            -  -      827 


IV.     THE  INFUSORIA. 

LXIII.  The  Infusoria. 

Introduction  and  general  methods  of  examination,  p.  829. 

Parasitic  species,        -        -        ...  .        .      830 


PART  VI. 

LXIV.  The  filtrable  viruses. 

Introduction,  -  -      835 

Section  I.     The  virus  of  Pleuro-pneumonia  in  cattle,      -  836 
(1)  Experimental  inoculation  and  vaccination,  p.  836.     (2)  Methods 
of  diagnosis  and  characteristics  of  the  organism,  p.  837. 

Section  II.     The  virus  of  Foot  and  Mouth  disease,  -      838 

Section  III.     The  virus  of  Horse-sickness,      -  838 

Section  IV.     The  virus  of  Rinderpest,    -  -      839 

Section  V.     The  virus  of  Bird  plague,  -  -       839 

Section  VI.     The  virus  of  Sheep-pox,  -       839 
The  "  infectious  epithelioses,"  p.  840. 

Section  VII.     The  virus  of  Cow-pox,  -      840 

Section  VIII.     The  virus  of  Yellow  fever,  841 

Section  IX.     The  virus  of  Rabies,  841 


xxx  CONTENTS 

Section  X.     Filtrable  viruses  in  the  Pasteur  elloses,  -       842 

Section  XI.     The  virus  of  Swine-fever,  -  843 

Section  XII.     The  virus  of  Acute  anterior  polio-myelitis,  -       844 

Section  XIII.     The  virus  of  Typhus  fever,    -  -       847 


PART  VII.     THE  APPLICATION  OF  BACTERIOLOGICAL  METHODS 
TO  THE  EXAMINATION  OF  WATER,   SEWAGE  AND  AIR. 

LXV.  The  bacteriological  examination  of  water. 

Introduction,  -  851 

'Section    I.     The    collection    and    transmission    of  samples    of 

water,      -  -       851 

Section  II.     The  methods  of  examination,  853 

(1)  Enumeration  of  the  organisms,  p.  853.  (2)  Determination  of  the 
nature  of  the  organisms  present,  p.  856.  (3)  Houston's  method  of 
water  examination,  p.  858. 

The  bacteriological  examination  of  sewage,  p.  861. 

LXVI.  The  bacteriological  examination  of  air. 

Introduction,  -  -       862 

1.  Original  methods,  p.  862.  2.  Methods  employed  at  the  present 
day,  p.  864. 

Index,  -      868 


PART  I. 
GENERAL  TECHNIQUE. 


CHAPTER  I. 
STERILIZATION. 

Introduction. ' 

Section  I. — Sterilization  by  dry  heat,  p.  4. 

1.  Sterilization  in  a  naked  flame,  p.  4.     2.   Sterilization  by  hot  air,  p.  4. 
Section  II. — Sterilization  by  moist  heat,  p.  7. 

1.  Sterilization   in    steam    at    100°  C.,    p.    7.       2.  Sterilization    in   steam    under 
pressure,  p.  9.     3.  Sterilization  by  discontinuous  heating,  p.    12. 
Section  III. — Sterilization  by  filtration,  p.  14. 

1.  The    filtration    of   water,    p.   15.     2.  The  filtration  of  culture  media,  p.  18  ; 
(A)  by  compression,  p.   18 ;    (B)  by  aspiration,  p.   19.     3.  The  filtration  of  small 
quantities  of  liquid,  p.  24. 
Section  IV. — Sterilization  by  antiseptics,  p.  26. 

FOB  the  study  of  any  given  micro-organism  it  is  necessary  to  have  a  pure 
culture  of  the  organism,  that  is  to  say  a  culture  from  which  all  other  organ- 
isms have  been  excluded.  Since  micro-organisms  are  universally  present 
in  air  and  water  and  in  the  ambient  media  generally,  it  is  essential  that  all 
vessels,  culture  media,  instruments,  etc.,  to  be  used  in  the  preparation  and 
investigation  of  pure  cultures  should  themselves  be  free  from  living  organisms, 
or  in  other  words  be  sterile.  Sterilization  therefore  means  the  destruction  of 
living  micro-organisms  in,  [or  their  removal  from,]  materials  and  apparatus 
used  in  bacteriological  investigations. 

It  would  however  be  useless  to  sterilize  vessels,  instruments  and  culture 
media  unless  steps  were  also  taken  to  prevent  them  from  again  becoming 
soiled  (using  the  word  in  its  bacteriological  sense)  before  being  put  to  their 
proper  use  ;  they  must  therefore  be  dealt  with  in  such  a  manner  that  when 
sterilized  they  are  completely  protected  from  contact  with  extraneous 
organisms. 

To  accomplish  this,  vessels  with  a  narrow  mouth  such  as  flasks,  bottles 
and  tubes  are  plugged  with  wool  after  being  washed  and  before  sterilization, 
such  articles  as  watch-glasses,  dishes,  etc.,  are  wrapped  in  paper,  [while  metal 
instruments,  pipettes,  etc.,  may  be  placed  in  a  metal  cylinder  or  box,  or  in 
a  piece  of  glass  tubing  of  large  diameter  plugged  with  wool  at  the  two 
ends], 

1.  Plugging  with  wool. — To  plug  a  narrow- mouthed  vessel  take  a  small  piece 
of  non-absorbent  cotton-wool,  fold  it  by  twisting  it  round  and  round,  insert  one 
end  into  the  mouth  of  the  vessel  and  then  force  it  gently  to  a  depth  of  2  to  3  cm. 
leaving  the  other  end  projecting  from  the  orifice.  It  is  better  that  the  plug  should 
be  too  large  than  too  small. 


STERILIZATION   BY   DRY   HEAT 


2.  Paper  covers. — For  wrapping  up  vessels  and  other  articles  ordinary  filter 
paper  may  be  used,  but  any  common  paper  of  decent  texture  is  equally  serviceable 
and  has  the  merit  of  being  more  economical. 

(a)  Watch-glasses,  Petri "dishes,  etc.,  should  be  wrapped  in  several  folds  of  paper. 

(6)  Wide-mouthed  cylindrical  or  conical 
vessels  only  need  to  have  the  opening 
covered  with  a  double  layer  of  paper, 
though  this  should  be  large  enough  to  allow 
of  it  being  turned  down  and  twisted  [or 
tied]  round  the  vessel,  so  that  the  greater 
part  of  the  latter  is  enveloped.  In  doing 
this,  be  careful  not  to  tear  the  paper, 
which  is  apt  to  split  on  the  edges  of  the 
opening. 

[3.  Other  methods. — Petri  dishes,  pip- 
ettes, watch-glasses,  metal  instruments, 
etc.,  may  be  conveniently  enclosed  in 
copper  boxes  of  suitable  shape,  which 
should  have  tightly  fitting  lids  with  a  deep 
overlap.  For  ordinary  Petri  dishes  a 
circular  copper  cylinder  25  x  12  cm.  (fig.  1) 
containing  a  moveable  tray  may  be  used  ; 
for  pipettes  a  similar  but  longer  and 
narrower  cylindrical  metal  vessel  or  rect- 
angular box  is  useful.  Pipettes  may  also 
be  enclosed  in  a  piece  of  large  glass  tubing,  which  is  then  plugged  at  both  ends 
with  wool.  The  pipettes  must  of  course  be  themselves  plugged  at  the  upper  end 
with  wool.] 

Sterilization  may  be  effected  in  one  of  several  ways,  the  most  generally 
employed  being  heat  and  filtration  ;  chemical  antiseptics  are  seldom  used  in 
bacteriology.  The  methods  of  sterilization  commonly  employed  will  now  be 
considered  in  detail. 


FIG.  1.— Copper  cylinder  with  deep  overlap 
in  which  to  sterilize  Petri  dishes. 


SECTION  I.— STERILIZATION  BY  DRY  HEAT. 

1.  Sterilization  in  a  naked  flame. 

1.  The  simplest  means  of  sterilizing  a  metal  instrument  is  to  heat  it  to 
redness  in  a  spirit  flame  or  Bunsen  burner.     This  method  is  always  adopted 
for  sterilizing  platinum  wires  and  iron  and  nickel  spatulas. 

Knives  and  similar  instruments  can  also  of  course  be  sterilized  by  heating 
them  in  a  flame,  but  on  account  of  the  injury  done  to  the  instrument  the  method 
is  very  rarely  adopted. 

An  instrument  which  has  been  sterilized  by  heating  to  redness  must  be 
cooled  before  it  is  allowed  to  touch  any  material  which  is  to  be  used  for 
sowing  cultures. 

2.  An  instrument  may  be  sterilized  by  flaming  it,  i.e.  by  passing  it  rapidly 
through  a  hot  flame. 

Only  pipettes,  glass  rods,  and  other  instruments  with  polished  surfaces 
devoid  of  crevices  in  which  organisms  might  escape  destruction  can  be 
sterilized  in  this  way,  so  that  the  method  is  of  limited  application. 

2.  Sterilization  by  hot  air. 

Exposure  to  hot  air  is  the  usual  method  of  sterilizing  all  glass  and  porcelain 
apparatus,  instruments  with  metal  handles,  etc.,  but  it  is  not  suitable  for 
organic  substances,  with  the  exception  of  wool  and  paper. 


HOT   AIR   STERILIZERS  5 

Some  form  of  apparatus  in  which  sterilization  can  be  effected  by  means 
of  hot  air  is  to  be  found  in  all  laboratories. 

To  ensure  efficient  sterilization,  the  temperature  must  be  maintained  at  approxi- 
mately 180°  C.  for  30  minutes.  Cotton-wool  and  paper  are  slightly  scorched  and 
browned  at  this  temperature. 

Hot  air  sterilizers. 

The  various  forms  of  hot  air  sterilizers  differ  from  one  another  only  in 
details  and  in  external  appearance,  the  principles  of  construction  and  methods 
of  use  being  the  same  in  all. 

1.  Pasteur's  sterilizer  (fig.  2)  is  a  double-walled  cylinder  of  sheet  iron 
with  a  chimney  outlet,  and  is  fitted  internally  with  a  wire  basket  in  which 
the  articles  to  be  sterilized  are  packed.  The  top  is  closed  by  a  lid,  through 


FIG.  2. -Pasteur's  hot  air  sterilizer. 

a  hole  in  which  a  cork  carrying  a  thermometer  registering  to  200°  C.  is 
passed.  The  heat  is  derived  from  a  large  gas  burner  below,  and  when  this 
is  lighted  the  heated  air  rising  from  the  bottom  of  the  stove  circulates  between 
the  inner  and  outer  walls  and  escapes  up  the  chimney. 

2.  Chantemesse's  and  PoupinePs  hot  air  sterilizers  are  rectangular  and 
cupboard-shaped.  They  are  fitted  internally  with  moveable  shelves  on  which 
the  glass  and  other  apparatus  is  arranged. 

[3.  Hearson's  hot  air  sterilizer  (fig.  3)  is  similar  in  shape  to  Chantemesse's, 
but  is  provided  with  an  arrangement  by  which  the  gas  is  automatically 
regulated  when  the  temperature  has  reached  the  point  for  which  the  regu- 
lator is  set.] 


6 


STERILIZATION   BY   DRY   HEAT 


Technique  of  sterilization  by  hot  air. 

(a)  Carefully  wash  and  rinse  in  a  large  volume  of  water  all  apparatus, 
whatever  its  nature,  until  all  traces  of  organic  matter  have  been  removed. 
Unless  the  cleansing  of  glass  for  example  be  very  thorough,  black  stains, 
due  to  the  charring  of  organic  matter  during  the  heating  process,  will  be 
found  on  the  surface  after  sterilization.  After  washing  allow  the  apparatus 
to  dry,  being  specially  careful  in  the  case  of  glass,  to  avoid  subsequent  break- 
age during  heating.  When  dry,  treat  each  article  in  the  manner  already 
described,  either  plugging  with  wool,  wrapping  in  paper,  or  packing  in  a 
metal  box,  according  to  its  nature  and  use. 


FIG.  3.— Hearson's  hot  air  sterilizer. 

(b)  Place  the  articles  in  the  sterilizer,  taking  care  that  neither  wool  nor 
paper  touch  the  floor  or  sides,  for  these  substances  will  char  if  they  come  in 
contact  with  the  heated  metal,  and  a  tarry  product  rich  in  antiseptic  sub- 
stances will  be  deposited  on  the  sterilized  vessels,  which  will  interfere  with 
the  subsequent  growth  of  organisms.     If  by  accident  charring  should  take 
place,  the  articles  which  have  been  soiled  must  be  washed,  first  in  alcohol, 
then  in  water,  dried  and  re-sterilized. 

To  avoid  charring  and  breakage,  it  is  advisable  to  place  one  or  two  fire 
bricks  on  the  bottom  of  the  sterilizer  to  keep  the  contents  from  touching  the 
heated  metal  surface. 

(c)  Close  the  sterilizer  and  place   the   thermometer  in  position,  pushing 
the  latter  well  down  into  the  interior. 


STERILIZATION   IN   STEAM   AT   100°  C.  7 

(d)  Light  the  gas.     It  is  well  to  hold  a  lighted  taper  to  the  burner  before 
turning  on  the  tap,  since  if  gas  escape  it  will  mix  with  the  air  between  the 
inner  and  outer  walls  of  the  sterilizer  and  so  tend  to  cause  an  explosion. 

(e)  Regulate  the  flame  so  that  the  temperature  rises  slowly  ;    this  is  par- 
ticularly important  if  the  sterilizer  contain  vessels  of  thick  glass,  e.g.  test- 
tubes  on  feet,  glass  dishes,  etc. 

(/)  When  the  thermometer  records  a  temperature  of  175°-180°  C.  in  the 
interior  of  the  sterilizer,  lower  the  gas  gently,  leaving  sufficient  flame  to 
maintain  the  temperature  at  180°  C.  or  thereabouts  for  half  an  hoar  or  so. 

With  a  little  practice  this  is  easily  done.  Rather  than  use  the  fingers  it  is  better 
to  manipulate  the  tap  by  tapping  it  with  some  heavy  instrument  such  as  the  spanner 
used  for  tightening  the  bolts  of  the  autoclave,  which  will  give  very  delicate  control 
over  the  supply  of  gas,  and  will  obviate  the  annoyance  caused  by  accidentally 
turning  out  the  gas  altogether. 

When  experience  has  been  gained,  a  thermometer  can  be  dispensed  with;  at 
a  temperature  of  180°  C.  wool  and  paper  become  slightly  scorched,  and  when 
this  effect  is  noted  the  gas  is  turned  down. 

(g)  When  sterilization  is  completed  turn  out  the  gas,  but  allow  the  tem- 
perature to  fall  considerably  before  removing  the  contents,  because  glass, 
and  especially  thick  glass,  is  liable  to  crack  if  exposed  to  a  sudden  change 
of  temperature. 

[With  Hearson's  hot  air  sterilizer  the  procedure  is  the  same,  except  that  stage 
(/)  is  omitted ;  when  the  temperature  for  which  the  capsule  is  set  is  reached,  the 
gas  is  automatically  lowered.  It  is  only  necessary  therefore  to  note  when  the 
thermometer  reaches  the  point  at  which  sterilization  is  to  be  effected,  and  half 
an  hour  later  to  turn  out  the  gas  and  proceed  as  in  (g).] 

SECTION  II.— STERILIZATION  BY  MOIST   HEAT. 

Sterilization  by  moist  heat  may  be  effected  in  one  of  three  ways. 

1.  By  heating  in  water  or  steam  at  100°  C. 

2.  By  heating  in  steam  under  pressure. 

3.  By  discontinuous  heating  at  low  temperatures. 

1.  Sterilization  in  steam  at  100°  C. 

Simple  boiling  or  exposure  to  steam  at  100°  C.,  even  though  the  exposure 
be  prolonged,  is  not  a  reliable  method  of  sterilization. 

When  micro-organisms  have  been  dried,  their  resistance  to  the  effects  of  heat 
is  much  enhanced,  and  especially  is  this  the  case  when  they  are  mixed  with  sub- 
stances of  an  albuminoid  nature.  Further  there  are  certain  resistant  forms  of 
bacterial  protoplasm  known  as  spores,  which  in  the  majority  of  cases  at  least 
are  not  destroyed  by  heating  to  100°  C.,  even  when  the  temperature  is  maintained 
for  several  minutes. 

In  France  sterilization  by  moist  heat  at  100°  C.  is  very  seldom  employed, 
except  for  sterilizing  syringes  for  inoculation.  In  this  case  a  sufficient 
degree  of  asepsis  is  obtained  by  boiling  in  water  for  15  to  20  minutes  at 
ordinary  atmospheric  pressure. 

[In  England  on  the  other  hand,  and]  in  Germany,  sterilization  by  moist 
heat  at  100°  C.  is  in  general  use.  The  operation  is  carried  out  in  a  Koch's 
sterilizer  or  steamer,  and  must  be  repeated  at  intervals  of  24  hours  on  at 
least  two,  but  ordinarily  on  three,  successive  days. 

This  method  is  the  outcome  of  an  observation  by  Tyndall  to  the  effect  that 
while  it  is  impossible  to  sterilize  an  infusion  of  hay  by  boiling  it  continuously  even 
for  a  prolonged  period,  yet  by  boiling  it  for  a  short  time  on  three  successive  days 
all  living  organisms  are  destroyed. 


8 


STERILIZATION   BY  MOIST  HEAT 


This  process  embodies  the  principle  of  sterilization  by  discontinuous  heating. 
The  explanation  put  forward  by  Tyndall  was  that  the  hay  infusion  contains  both 
bacilli  and  spores  (B.  subtilis).  By  heating  to  100°  C.  the  bacilli,  but  not  the 
spores,  are  killed.  The  latter  germinate  as  the  fluid  cools,  and  are  killed  during  the 
second  heating.  A  few  spores  however  escape  destruction  on  the  second  heating  ; 
these  will  have  germinated  by  the  time  the  third  heating  is  due.  After  the  third 
heating  then  sterilization  is  completed.  The  explanation  now  given  however  is 
that  the  resistance  of  micro-organisms  is  gradually  lowered  under  the  influence  of 
repeated  heating. 

Steamers. 

1.  Koch's  steamer. — Koch's  steamer  (fig.  4)  consists  of  a  cylindrical 
copper  boiler,  provided  with  a  water  gauge  below  and  closed  above  by  a  lid 
through  a  hole  in  which  a  thermometer  can  be  passed.  It  is  fitted  with 

perforated  and  moveable  metal 
trays  on  which  to  rest  the  appar- 
atus. 

A  metal  cylinder  open  at  both 
ends  is  often  supplied  with  the 
sterilizer,  so  that  the  latter  can 
be  lengthened  when  necessary  by 
fitting  the  metal  cylinder  on  top. 
Technique.  —  When  sterilizing 
culture  media  by  steam  at  100°  C., 
it  is  advisable  to  use  vessels  already 
sterilized  in  the  hot  air  sterilizer. 
(a)  Pour  sufficient  water  into 
the  steamer  to  reach  the  level 
marked  on  the  water  gauge.  Stand 
the  vessels  on  the  trays,  and  if 
extra  space  be  needed  adjust  the 
lengthening  cylinder.  Put  on  the 
lid,  and  insert  the  thermometer. 

(6)  Light  the  gas  under  the 
boiler,  note  when  steam  begins  to 
escape  from  under  the  lid — the 
thermometer  will  then  register 
98°-100°  C.— and  maintain  the 
apparatus  at  this  temperature  for 
30  minutes. 

(c)  Heat  again  in  a  similar  manner  on  the  two  following  days. 
When  the  flasks,  tubes,  etc.,  are  taken  out  of  the  steamer,  the  wool  plugs 
are  generally  wet  with  water  of  condensation ;  and  since  wool  is  only  efficient 
as  a  filter  for  micro-organisms  so  long  as  it  is  absolutely  free  from  moisture, 
the  vessels  may  be  put  in  the  incubator  for  an  hour  or  two  to  dry  the 
plugs. 

In  many  of  the  newer  patterns  of  steamers  the  steam  circulates  between 
double  walls  before  escaping,  thus  maintaining  an  absolutely  constant  tem- 
perature in  the  steamer.  Some  forms  are  further  provided  with  a  constant- 
level  adjustment.  [One  of  the  most  useful  of  these  newer  patterns  is  that 
made  by  Hearson.] 

[2.  Hearson's  steamer. — Hearson's  steamer  (fig.  5)  consists  of  two  copper 
cylinders,  one  suspended  within  the  other,  thus  conserving  the  heat.  By 
means  of  a  special  regulator  the  gas  is  automatically  lowered  when  the  inner 
chamber  is  full  of  steam,  and  this,  instead  of  escaping  into  the  sterilizing 


FIG.  4.— Koch's  steamer. 


STERILIZATION   IN   STEAM   UNDER   PRESSURE  9 

room,   is  condensed  and  returned  to  the  boiler.     A  further  advantage  is 
that  the  water  is  added  from  the  outside.] 

2.  Sterilization  in  steam  under  pressure. 

Water,  syringes,  india-rubber  apparatus,  niters,  etc.,  are  generally  steri- 
lized by  heating  in  steam  under  pressure.  This  method  is  also  in  general 
use  for  the  sterilization  of  certain  culture  media,  but  is  not  particularly 
suitable  for  steel  cutting  instruments,  as  it  destroys  the  edge. 


FIG.  5.— Hearson's  steamer. 


FIG.  6. — Chamberland's  autoclave. 


Exposure  to  steam  at  a  temperature  of  115°  C.  for  20  minutes  is  in  most 
cases  sufficient  to  ensure  sterilization,  but  some  media,  potato  for  example, 
require  a  temperature  of  120°  C. 

Some  of  the  commoner  forms  of  autoclave  may  be  shortly  described  here. 

Autoclaves. 

1.  Chamberland's  autoclave  (fig.  6). — This  autoclave  consists  of  a  cylindrical 
copper  boiler,  the  free  edge  of  which  is  turned  out  flange  wise.  A  flanged 
bronze  cover  is  secured  to  this  edge  by  screw  bolts,  and  the  whole  is  made 
air-tight  by  the  insertion  of  an  india-rubber  washer  between  the  two  metal 
flanges. 

The  cover  is  provided  with  a  safety  valve,  a  steam  tap,  and  a  manometer 
which  records  the  pressure  in  atmospheres  and  the  temperature  in  degrees 
centigrade.  The  boiler  contains  a  removeable  copper- wire  basket,  which 
rests  on  short  feet  (5-6  cm.)  on  the  bottom  of  the  boiler.  The  boiler 


10  STERILIZATION   BY   MOIST   HEAT 

itself  is  supported  within  a  cylindrical  sheet-iron  or  copper  furnace  provided 
with  one  or  two  rings  of  Bunsen  burners. 

Technique. — (a)  Pour  sufficient  water  into  the  boiler  to  reach  to  just 
below  the  bottom  of  the  wire  basket ;  distilled  water  is  preferable,  as  by 
its  use  furring  is  avoided. 

Place  the  apparatus  to  be  sterilized  in  the  basket,  and  lay  two  or  three 
thicknesses  of  cloth  or  paper  over  the  wool  plugs  to  prevent  condensation 
water  dropping  on  to  them  from  the  cover. 

(b)  Adjust  the  india-rubber  washer,  put  on  the  cover,  and  screw  up  the 
bolts  with  the  fingers.     It  is  better  to  use  the  fingers  than  the  key  provided 
with  the  autoclave,  because  with  the  latter  an  unnecessary  amount  of  force 
is  very  likely  to  be  applied,  with  the  result  that  the  washer  is  quickly  ruined  ; 
moreover,  careless  manipulation  with  the  key  will  soon  strip  the  screws. 

(When  the  autoclave  is  not  in  use,  the  bolts  should  remain  loosened,  and  the 
washer  removed  and  hung  up  because  if  left  under  the  cover  it  gets  crushed.) 

(c)  Open  the  steam  tap. 

(d)  It  will  be  sufficient  to  light  one  ring  of  burners.     Hold  the  taper  to  the 
burner  before  turning  on  the  gas,  and  take  special  notice  that  the  burners 
do  not  light  below  :   should  this  happen,  turn  out  the  gas  and  re-light  it. 

(e)  As  soon  as  the  water  begins  to  boil,  steam  will  escape  from  the  tap 
in  the  cover,  and  must  be  allowed  to  continue  to  do  so  until  the  pressure  of 
the  steam  within  causes  it  to  issue  with  a  whistling  sound  in  a  powerful  and 
continuous  jet. 

The  object  of  this  manoeuvre  is  to  expel  the  whole  of  the  air  from  the  interior 
of  the  autoclave,  since  if  any  air  remain  in  the  boiler  the  manometer  readings  will 
not  be  reliable.  Still  however  much  care  be  taken  it  is  impossible  to  drive  out 
all  the  air,  and  the  larger  the  autoclave  the  larger  will  be  the  volume  of  air  remaining. 
A  more  effectual  means  of  expelling  the  air  is  to  compress  and  decompress  repeatedly 
by  opening  and  shutting  the  steam  tap,  but  this  method  should  never  be  adopted 
when  sterilizing  fluids  because  under  the  influence  of  a  sudden  lowering  of  the 
pressure  the  plugs  and  contents  of  the  flasks  and  tubes  are  driven  out  by  the  violent 
boiling  of  the  liquids. 

Now  close  the  steam  tap.  The  pressure  and  temperature  will  rise  rapidly, 
and  when  the  manometer  records  the  temperature  required  (115°-120°  C.), 
lower  the  gas  and  regulate  it  by  trial  until  the  manometer  reading  is  steady. 
Continue  the  heating  at  this  temperature  for  20  minutes. 

(/)  When  sterilization  is  completed,  turn  out  the  gas ;  the  manometer 
needle  soon  falls  to  zero,  and  then,  but  not  until  then,  open  the  steam  tap. 
When  all  the  steam  has  escaped  unscrew  the  bolts,  raise  the  cover,  and  remove 
the  contents.  If  the  plugs  be  damp  it  is  well  to  put  the  flasks,  etc.,  in  the 
incubator  until  the  wool  dries. 

The  following  minor  practical  details  in  the  working  of  an  autoclave  may  be 
mentioned.  It  is  important  never  to  open  the  steam  tap  until  the  pressure  within 
the  apparatus  has  fallen  to  the  zero  mark  on  the  manometer,  for  the  reason  already 
given,  namely  that  under  the  influence  of  sudden  decompression  the  fluid  contents 
of  the  flasks,  etc.,  are  liable  to  be  discharged  into  the  autoclave.  Again,  to  avoid 
accidents  by  scalding  from  an  escape  of  steam  beneath  the  cover,  the  steam 
tap  must  always  be  opened  before  the  bolts  are  loosened.  Lastly,  to  obviate  any 
difficulty  in  lifting  the  cover  owing  to  the  rubber  washer  sticking  to  the  metal, 
always  open  the  autoclave  before  the  latter  gets  quite  cold. 

Note. — The  autoclave  is  also  available  for  sterilization  at  100°  C.  The 
procedure  will  be  the  same  as  regards  the  first  four  steps  a,  b,  c,  d,  but  the 
steam  tap  must  be  left  open  the  whole  time  (30  minutes),  and  the  gas 
burners  regulated  so  that  the  pressure  as  recorded  by  the  manometer  needle 
does  not  rise  above  the  zero  point. 


AUTOCLAVES 


11 


It  is  obvious  of  course  that  a  sufficient  quantity  of  water  must  be  put 
into  the  boiler  before  commencing  the  sterilization.  Heating  should  never 
be  continued  for  longer  than  30  to  40  minutes  in  case  the  boiler  should 
boil  dry. 

2.  Ducretet  and  Lejeune's  autoclave. — The  principle  and  working  of  the 
instrument  are  the  same  as  in  the  case  of  Chamberland's  autoclave.     The 
tall  form  of  boiler  however  makes  it  especially  useful  for  the  sterilization 
of  long  pieces  of  apparatus  and  of  porcelain  filter  bougies  ;  as  many  as  thirty 
of  the  latter  can  be  accommodated  at  one  and  the  same  time  by  means  of  a 
special  pattern  of  support.     The  autoclave  will  withstand  a   pressure   of 
3  or  4  atmospheres,  and  is  strong  enough  to  be  used  for  sterilization  by 
means  of  compressed  carbonic  acid  (d' Arson val). 

To  facilitate  manipulation,  some  minor  alterations  have  been  introduced  in  the 
construction  of  the  newest  models  of  autoclaves.  For  instance,  in  one  made  by 
Adnet  the  cover  is  secured  by  a  gearing  controlled  by  a  single  screw  instead  of  by 
bolts.  In  another,  made  by  Rongier,  the  cover  is  fitted  with  an  hinge,  and  in  yet 
another,  by  Radias,  with  a  lever. 

3.  Vaillard    and    Besson's    autoclave. — In   large    laboratories   where,    for 
instance,  toxins  for  immunizing  horses  in  the  preparation  of  therapeutic 
serums  or  for  other  purposes  are  required  in 

large  bulk,  and  the  consumption  of  media  is 
considerable,  it  is  necessary  or  at  least  con- 
venient to  have  some  more  commodious  form 
of  autoclave  than  Chamberland's.  In  such 
cases  Vaillard  and  Besson's  pattern  is  available 
(%.  7). 

This  autoclave l  consists  of  a  large  cylindrical  boiler 
with  double  walls.  The  apparatus  to* be  sterilized  is 
arranged  on  shelves  in  a  central  space.  The  steam 
rising  from  the  water  in  the  double  bottom  ascends 
between  the  inner  and  outer  walls,  passes  through  the 
sterilizing  chamber  from  above  downwards,  and  escapes 
through  a  safety  valve,  the  escape  being  regulated  in 
such  a  .manner  that  the  pressure  and  therefore  the  tem- 
perature rise  gradually.  When  the  temperature  reaches 
115°  C.,  the  safety  valve  automatically  allows  the  steam 
to  escape  sufficiently  to  prevent  any  further  increase 
of  pressure.  The  boiler  is  also  fitted  with  a  lateral 
funnel  through  which  the  water  may  be  poured  in,  a 
tap  by  which  the  level  of  the  water  is  regulated,  a 
manometer  and  a  safety  valve.  The  construction  of 

this  apparatus  is  such  that  sterilization  is  effected  in  a  current  of  steam,  and  a  further 
advantage  is  that  all  the  air  is  expelled  without  resort  to  decompression,  the  disadvan- 
tages of  which  have  already  been  noted. 

Technique. — (a)  Place  the  apparatus  in  the  chamber  S,  and  secure  the  cover  firmly 
by  means  of  the  screw  bolts.  (6)  Open  the  tap  of  the  lateral  supply  funnel  and  pour 
in  water  until  it  runs  out  at  P,  which  must  also  have  been  previously  opened ;  then  close 
both  taps  and  raise  the  valve  D.  (c)  Light  the  stove.  (In  France  charcoal  is  generally 
used  as  the  source  of  heat,  but  the  autoclave  is  also  constructed  to  work  with  gas.)  (d)  As 
soon  as  the  water  boils,  steam  will  rise  between  the  inner  and  outer  walls,  enter  the  steriliz- 
ing chamber,  and  escape  by  way  of  the  tube  leading  to  D.  When  the  pressure  is  sufficient 
to  cause  the  steam  to  issue  in  a  powerful  jet,  lower  the  valve  D.  The  temperature  and 
pressure  within  the  autoclave  will  now  rise,  and  will  be  registered  on  the  manometer  M. 
The  steam  escapes  more  and  more  violently  as  the  pressure  increases,  until  the  tem- 
perature for  which  the  valve  has  been  regulated  (usually  115°  C.)  is  reached,  when 
the  volume  of  escaping  steam  is  such  as  to  prevent  any  further  rise  of  temperature.  The 

1  Annalea  de  V  Institut  Pasteur,  1894. 


FIG.  7.— Vaillard  and  Besson's 
autoclave. 


12  STERILIZATION   BY   MOIST   HEAT 

temperature  and  pressure  must  be  maintained  for  20  minutes,  reckoning  from  the  moment 
when  it  reaches  115°  C.  (e)  When  the  necessary  time  has  elapsed,  remove  the  source  of 
heat  and  allow  the  autoclave  to  cool  until  the  pressure  reaches  zero  on  the  manometer, 
then  open  the  steam-tap  (not  shown  in  the  illustration),  and  raise  the  cover. 

Note. — The  autoclave  may  also  be  used  for  sterilization  at  100°  0.  The  technique  is 
the  same  as  in  the  preceding  case,  except  that  the  valve  D  is  never  raised.  Under  these 
conditions  steam  will  issue  in  a  powerful  jet  during  the  whole  operation.  The  tem- 
perature must  be  maintained  for  30  minutes  after  reaching  100°  C. 

Sterilization  can  also  be  effected  at  any  temperature  between  100°  and  115°  C.  by 
suitably  altering  the  position  of  the  knobbed  handle  of  the  valve ;  the  further  the  handle 
is  fronTthe  vertical  the  less  will  the  temperature  rise  above  100°  C. 

Method  of  controlling  the  temperatures  at  which  sterilization  was  effected. 

In  laboratories  where  the  sterilization  of  apparatus,  etc.,  is  entrusted  to  laboratory 
assistants,  it  is  convenient  to  have  a  method  of  controlling  the  temperature  at  which 
sterilization  was  effected.  This  may  be  done  by  placing  a  maximum  thermometer 
or,  more  conveniently,  a  fragment  of  fusible  alloy  or  some  chemical  compound 
of  suitable  melting  point  (110°-120°  C.)  alongside  the  apparatus  in  the  autoclave. 
If  a  powder  be  used  it  may  be  mixed,  as  suggested  by  Demandre,  with  a  trace  of 
some  dye,  and  sealed  up  in  a  small  glass  ampoule.  The  small  amount  of  dye  used 
is  not  visible  in  the  powder,  but  when  the  latter  melts  the  dye  diffuses  through 
it,  and  on  cooling  forms  a  coloured  bead. 

The  following  substances  are  suitable  for  the  purpose,  the  temperature  in  brackets 
indicating  the  melting  point:  benzonaphthol  (110°C.);  antipyrine  and  sulphur 
(113°  C.);  resorcin  (119°  C.)  ;  benzoic  acid  (121°  C.). 

For  coloured  beads  the  following  formulae  may  be  employed : 

Melting  at  110°  C.  Benzonaphthol,  -         100  grams. 

Safranin,    -  O'Ol  gram. 

Melting  at  121°  C.  Benzoic  acid,      -  100  grams. 

Brilliant  green,  -  O'Ol  gram. 

3.  Sterilization  by  discontinuous  heating  at  low 
temperatures. 

Some  substances  used  as  culture  media,  being  rich  in  albumin,  cannot  be 
heated  to  boiling  point  without  marked  alteration  and  to  some  extent 
destruction  of  their  properties.  Serum  is  a  case  in  point. 

Pasteur  showed  that  such  media  can  be  better  sterilized  by  heating  them 
at  a  low  temperature  (55°-60°  C.)  for  a  long  time  than  at  a  high  tempera- 
ture for  a  short  time.  This  prolonged  heating  at  a  low  temperature  con- 
stitutes Pasteurization.  In  practice  however  it  is  found  that  to  be  effectual, 
pasteurization  must  be  combined  with  the  method  of  discontinuous  heating- 
devised  by  Tyndall  (p.  7). 

Technique. — Distribute  the  medium  into  a  series  of  sterile  flasks  with 
long  necks  (fig.  35,  p.  46),  each  flask  being  about  three-fourths  filled,  and 
seal  the  mouths  in  a  blow-pipe.  [Flasks  and  test-tubes  covered  with  india- 
rubber  caps  (p.  29)  over  the  wool  plugs  can  be  used  equally  well.] 

Place  the  flasks  in  a  water  bath  fitted  with  a  thermometer,  slowly  raise 
the  temperature  and  regulate  the  gas  flame  so  that  it  remains  constant  at 
56°-57°  C.  for  an  hour,  then  turn  out  the  gas,  but  leave  the  flasks  in  the 
bath  until  they  are  quite  cool. 

The  flasks  must  be  heated  in  the  same  way  daily  for  a  week  before  the 
contents  can  be  regarded  as  sterile ;  and  even  then  they  ought  to  be  incu- 
bated at  37°  C.  for  two  or  three  days,  and  any  flask  in  which  a  growth  appears 
must,  of  course,  be  rejected. 

Water  baths. 

Conducted  in  the  manner  described,  this  method  of  sterilization  is  tedious, 
and  it  is  difficult  to  avoid  exceeding  a  temperature  of  58°  C.,  with  the  result 
that  the  albumin  coagulates,  rendering  the  medium  useless  for  the  purpose 


STERILIZATION   BY  DISCONTINUOUS   HEATING          13 

for  which  it  is  required.  Hence  it  is  better  to  have  some  form  of  water 
bath,  in  which  the  temperature  is  automatically  controlled  by  a  regulator 
on  the  gas  supply. 

[1.  Hearson's  water  bath. — This  is  a  very  convenient  and  reliable  form 
of  water  bath.  It  consists  of  a  cylindrical  copper  vessel  (fig.  8)  heated 
below  by  an  ordinary  fish-tail  gas  burner,  the  temperature  being  controlled 


FIG.  8.— Hearson's  water  bath. 


by  a  capsule  attached  to  the  outside  of  the  bath,  and  through  which  the 
gas  passes.  The  capsule  has  a  range  of  about  10°  C.,  and  within  these  limits 
the  temperature  is  regulated  by  means  of  a  milled  screw. 

[Technique. — Pour  sufficient  water  into  the  bath  to  reach  above  the  level 
of  the  regulator  outside.  Put  on  the  lid,  and  pass  a  thermometer  through 
the  hole  in  it,  being  careful  to  see  that  the  bulb  is  in  the  water.  Light  the 
gas.  The  temperature  gradually  rises  until  it  reaches  the  point  for  which 
the  regulator  is  set :  the  gas  is  then  automatically  lowered  and  the  tem- 
perature remains  stationary.  To  raise  the  temperature,  turn  the  screw 
clockwise,  to  lower  it,  contra-clockwise. 

[Note.— It  must  be  remembered  that  the  capsule  has  a  working  limit  of 
about  10°  only,  the  exact  limits  being  indicated  when  the  instrument  is 
supplied.  Consequently,  if  a  bath  is  required  to  work  sometimes  at 
55°-65°  C.,  and  at  other  times  at  75°— 85°  C.,  it  is  necessary  either  to  have 
two  baths,  or  a  single  bath  in  which  capsules  are  interchangeable. 

[Be  careful  always  to  see  that  the  water  is  above  the  level  of  the  top  of 
the  capsule,  and  when  filling  the  bath  never  add  water  of  a  temperature 
higher  than  that  for  which  the  capsule  is  regulated.] 

2.  Weissnegg's  water  bath. — Another  form  of  bath,  which  is  shown  in  fig.  9,  consists 
of  a  metal  vessel  fitted  with  a  Roux's  regulator  placed  in  a  side  chamber,  the  heat 
being  supplied  by  a  gas  burner  below. 


14 


STERILIZATION   BY   FILTRATION 


Technique. — Fill  the  vessel  about  three-fourths  full  of  water  and  immerse  the  flasks 
by  means  of  the  wire  tray,  put  on  the  lid,  pass  a  thermometer  through  the  opening  pro- 
vided for  the  purpose,  and  light  the  gas.  Watch  the  thermometer  carefully  until  the 
desired  temperature  is  reached,  then  set  the  regulator  in  the  manner  to  be  described  later 
(Chap.  IV.),  and  no  further  supervision  is  required. 

The  regulator  being  once  set  for  a  given  temperature  will  always  work  automatically 
at  that  temperature  until  it  is  again  altered,  so  that  beyond  lighting  the  gas  and  when 
necessary  pouring  water  into  the  bath  no  further  manipulation  is  required. 


FIG.  9.— Weissnegg's  water  bath. 

Note. — When  using  the  bath  for  the  first  time,  it  is  advisable  to  set  the  regulator  before- 
hand by  means  of  a  blank  experiment,  thus  avoiding  accidental  overheating  of  the 
medium.  Sterilization  is  then  carried  out  as  already  described,  the  medium  being 
heated  on  six  or  eight  consecutive  days  for  an  hour  each  time. 


SECTION   III.— STERILIZATION   BY  FILTRATION. 

The  application  of  heat  in  some  form  is  the  usual  method  of  sterilization 
used  in  bacteriological  work,  but  it  sometimes  happens  that  fluids  have 
to  be  dealt  with  which  cannot  be  subjected  to  even  a  moderate  degree  of 
heat  without  profoundly  altering  their  nature.  In  order  to  sterilize  such 
a  fluid,  it  is  passed  through  a  solid  bougie,  the  pores  of  which  are  so  fine 
that  while  liquids  and  solids  in  solution  pass  through,  micro-organisms  are 
retained.  Pasteur  in  his  early  work  utilized  plaster  plates  as  the  filtering 
medium,  but  as  a  result  of  Chamberland's  researches  porous  porcelain 
superseded  plaster. 

Filters. 

The  Pasteur- Chamberland  filter  consists  of  a  porous  porcelain  tube  or 
bougie  closed  at  one  end  but  open  at  the  other,  and  finished  at  the  latter  with 
a  nozzle  of  glazed  porcelain.  The  unfiltered  liquid  traverses  the  pores  of  the 
bougie  from  without  inwards,  and  issues  from  the  nozzle  filtered  and  steri- 
lized. The  Pasteur-Chamberland  bougies  are  made  in  two  grades  of  porosity. 


FILTRATION   OF   WATER 


15 


That  known  as  the  Chamberland  "  F  "  is  the  more  permeable, 
one  generally  used  both  for  .domestic  purposes  and  for  ordinary 
by  aspiration.  The  less  porous  and  harder  bougie,  the  Cham- 
berland "  B,"  is  only  used  for  nitration  under  pressure  (vide 
infra),  and  when  manipulating  fluids  containing  exceedingly 
minute  organisms,  e.g.  the  organisms  of  foot  and  mouth  disease, 
pleuro-pneumonia, horse-sickness,  etc.  (vide  "Filtrable  Viruses" 
Chap. LXIV.),  which  pass  through  the  more  porous  "F"  bougies. 

In  addition  to  the  Chamberland  bougies  there  are  other 
niters  of  a  similar  nature.  [The  Doulton  white-porcelain  filter 
(fig.  10)  has  been  found  to  be  "  at  least  as  efficient  in  the 
retention  of  micro-organisms  as  the  best  material  on  the  market, 
viz.  the  Pasteur-Chamberland  filter,"  and  to  "  excel  the  latter 
in  its  rate  of  filtration."  x] 

Another  filter,  Garros',  is  made  of  infusorial  earth.  This, 
[like  Doulton's]  filter,  has  all  the  essential  properties  of  a 
Chamberland  filter,  and  both  are  used  in  exactly  the  same 
way.  The  Berkefeld  bougie  is  also  made  of  infusorial  earth  ; 
it  is  inferior  to  the  Chamberland  "  B  "  in  that  it  wears  out 
more  rapidly  and  does  not  hold  back  the  smallest  organisms ; 
on  the  other  hand  it  filters  more  quickly  than,  and  does  not 
retain  dissolved  organic  matter  to  the  same  extent  as,  the 
Chamberland  filters.  For  the  latter  reason  it  is  especially 
useful  for  the  filtration  of  albuminous  fluids.  [But  it  must  be 
pointed  out  that  recent  experiments  have  shown  that  the 
Berkefeld  is  not  a  trustworthy  filter.2] 

There  are  several  ways  in  which  these  unglazed  porcelain 
and  similar  filters  may  be  used. 


and   the 
filtration 


FIG.  10.—  Doul- 
ton's porous  por- 
celain filter  with 
nozzle. 


1.  Filtration  of  water. 

Every    laboratory    has     a    filter 
attached,  to   a   water  tap,    for   the 
purpose    of   readily  obtaining  a  supply  of   sterile 
water  (fig.  11). 

The  filter  (Chamberland  F  [or  Doulton  white])  is 
contained  within  a  metal  cylinder,  through  the 
lower  end  of  which  it  is  inserted,  and  then  securely 
fixed  by  means  of  a  metal  screw-cap,  an  india- 
rubber  washer  intervening  ;  both  the  washer  and 
metal  cap  are  perforated  to  allow  the  passage  of 
the  glazed  nozzle.  The  upper  end  of  the  metal 
cylinder  is  screwed  on  to  a  tap  connected  with  the 
water  main.  When  the  tap  is  turned  on,  water 
runs  into  the  space  between  the  cylinder  and  the 
bougie,  traverses  the  bougie,  on  the  surface  of 
which  the  solid  matter  in  suspension  is  deposited, 
filter  attached  to  the  water  enterg  the  centraj  cavity,  and  escapes  from  the 

is  shown   by  the   dotted  lines     m0uth  of  the  glazed  nozzle, 
and  the  glazed  nozzle  is  seen 

projecting  below.  Preparation  of  the  filter. — 1.  Before  putting  a  filter 

into  the  metal  cylinder   it  is   absolutely  necessary  to 

ascertain  that  it  has  no  fissure  or  flaw  in  its  substance,  because  unless  it  be  perfect 
micro-organisms  will  quickly  find  their  way  through  it.     To  determine  whether  or 


FIG.  11.— A  porous  porcelain 

ter    attached    to    the    water 

main :  the  outline  of  the  filter 


^Journal  of  Hygiene.  1906,  1909.] 


[2  Journal  of  Hygiene,  1908,  1909.] 


16 


STERILIZATION   BY   FILTRATION 


no  the  bougie  is  sound,  attach  an  india-rubber  syringe  to  the  nozzle  and 
immerse  all  but  the  nozzle  in  a  cylinder  filled  with  water  (fig.  12).  By  squeezing 
the  syringe  air  will  be  driven  into  the  bougie,  and  if  a  fissure  be  present,  even 

one  so  small  as  to  be  invisible 
to  the  naked  eye,  bubbles  of  air 
will  stream  out  through  it  into 
the  water  and  will  at  once  ren- 
der it  apparent.  All  defective 
bougies  must  necessarily  be 
rejected. 

2.  The  filter  must   then    be 
sterilized.      After    testing    the 
bougie  and  while  it  is  still  wet, 
plug  the  nozzle  with  dry  wool 
and  sterilize  in  the  autoclave 
at  115°-120°C.  for  20  minutes. 
Fix  the  bougie   in   the   metal 
cylinder,    withdraw    the    wool 
plug,  and  the  filter  is  ready  for 
use. 

3.  Before  drawing  off  sterile 
water,    flame   the   nozzle  well 
with  a  spirit  flame. 

Cleansing  and  renovation  of  bougies. — 1.  When  in  use  the  external  surface  of  a 
filter  soon  gets  soiled,  and  organisms  are  then  likely  to  find  their  way  through  the 
pores.  It  is  necessary  therefore  that  filters  be  frequently  taken  out  and  cleaned  by 
scrubbing  with  a  stiff  brush  in  a  stream  of  running  water,  and  re- sterilized. 


FIG.  12.-  Method  of  testing  a  porcelain  filter. 


FIG.  13.— A  muffle  furnace. 


2.  But  this  surface  cleansing  does  not  prevent  the  pores  of  the  filter  becoming 
choked  after  a  time,  filtration  being  impeded  in  consequence  ;  when  this  occurs  a 
porcelain  filter  can  be  renovated  by  one  or  other  of  the  following  methods. 

(a)  After  scrubbing  the  filter  autoclave  it  at  120°  C.,  but  before  taking  it  out 


PREPARATION   OF   FILTER 


17 


of  the  autoclave  compress  and  decompress  several  times.  As  far  as  it  goes  this 
is  an  excellent  method  for  unchoking  a  filter  because  there  is  no  risk  of  damaging 
its  structure,  but  the  regeneration  is  only  partial. 

(&)  Clean  the  filter  as  above,  and  dry  it  thoroughly :  then  heat  it  to  redness  in 
a  Bunsen  flame.  This  involves  considerable  risk  of  fissuring  the  filter. 

(c)  The  best  method  is  to  heat  the  bougie  to  redness  in  an  incinerator  [or  "  muffle  " 
furnace]  (fig.  13). 

Note. — After  regenerating  a  filter  by  either  of  the  two  last  methods  it  must  be 
re-tested  to  make  certain  that  it  has  suffered  no  damage. 


FIG.  14.— Filtration  by  compression  (Gay-Lussac  pump). 

(d)  Lastly,  a  filter  may  be  regenerated  by  passing  through  it  a  0'5  per  cent, 
solution  of  potassium  permanganate,  followed  by  a  5  per  cent,  solution  of  sodium 
bisulphite  (Guinochet).  The  method  is  less  satisfactory  than  the  foregoing. 

3.  Whenever  culture  media  containing  micro-organisms  have  been  passed  through 
a  filter,  the  latter  must  be  autoclaved  immediately. 

B 


18 


STERILIZATION   BY   FILTRATION 


Berkefeld  bougie. — The  Berkefeld  bougie  does  not  lend  itself  to  heating  in  a 
flame  or  incinerator.  To  clean  it,  it  must  be  brushed  with  a  stiff  brush  in  a 
solution  of  sodium  carbonate,  washed  in  running  water,  and  then  autoclaved.1 


2.  Filtration  of  culture  media. 

Bougies  are  also  used  for  rendering  sterile  media  which  are  to  be  used 
for  growing  cultures,  and  for  freeing  a  culture  medium  of  the  organisms 
which  have  been  grown  in  it. 

This  may  be  effected  in  one  of  many  ways ;  but  nitration  should  always 
be  carried  out  under  pressure  either  by  putting  pressure  upon  the  liquid 
to  be  sterilized,  or  by  aspirating  the  nitrate  at  the  mouth  of  the  bougie. 

A.  Filtration  by  compression. 

The  original  method  was  to  pour  the  unfiltered  liquid  into  a  copper  reservoir 
A  (fig.  14),  and  then  to  force  it  through  the  filter  K  by  means  of  a  Gay- 
Lussac  pump  P. 

Technique. — Close  the  tap  G,  and  half  fill  the  reservoir  A  by  pouring  in  the  liquid 
through  the  opening  D.  Close  D  by  screwing  on  the  cap,  and  compress  the  air  in 
A  by  working  the  pump  P.  The  pressure  can  be  read  on  the  manometer  F.  When 
the  necessary  pressure  has  been  attained  (2  or  3  atmospheres  is  generally  sufficient), 
close  the  tap  E  and  slowly  open  G.  This  allows  the  liquid  to  pass  into  the  filtering 
chamber  H,  which  contains  a  sterile  Chamberland  filter  K  (size  B)  fitted  up  as 

described  above.     The  liquid  is  forced  through 
V  the  filter  and  issues  at  the  nozzle  where  it  can 

be  collected  aseptically. 

Collection  of  the  filtrate.— 1.  [A  Cobbett's 
bulb  is  a  useful  piece  of  apparatus  with  which 
to  collect  and  distribute  the  filtrate.  The 
illustration  (fig.  15)  shows  the  bulb,  which 
usually  has  a  capacity  of  about  200  c.c.  To 
render  it  available  for  the  present  purpose, 
plug  the  small  bulb  V  with  wool,  attach 
W  to  the  nozzle  of  the  filter  K  by  means  of 
stout  red  rubber  pressure-tubing,  and  with 
another  piece  of  rubber  tubing  connect  X  with 
a  short  length  of  glass  tubing  Z,  the  other  end 
of  which  has  been  drawn  out  to  a  fairly  narrow 
opening.  Select  a  small  india-rubber  plug  Y  with 
one  perforation,  slip  it  over  the  lower  end  of  Z 
and  push  it  up  until  it  fits  tightly  round  the 
tube,  then  enclose  the  lower  end  of  Z  in  a  test- 
tube  the  mouth  of  which  must  be  of  suitable  size 
to  fit  the  rubber  plug  Y.  After  thus  fitting  up 
the  filter  and  bulb,  autoclave  at  120°  C. 

[When  required  for  use,  fit  the  filter  K  in  its 
metal  case  H  (fig.  14),  and  screw  on  the  cap  L 
firmly.  Support  the  bulb  by  clamping  it  to  a 
retort  stand.  Clip  the  tubing  between  X  and  Z. 

[The  filtered  liquid  will  be  forced  into  the 
bulb,  the  rate  being  regulated  by  the  tap  G,  and  when  nearly  filled  turn  off  the  tap 
G ;  take  off  the  test-tube,  and  by  releasing  the  clip  between  X  and  Z  the  fluid  can 

f1  According  to  Dr.  Andrew  Wilson,  however,  it  would  appear  that  Berkefeld  bougies 
must  not  be  autoclaved.  "It  is  a  well-known  fact  that  in  consequence  of  the  composi- 
tion and  the  mounting  of  the  Berkefeld  filtering  cylinders,  they  do  not  stand  sterilization 
in  an  autoclave  at  120°  C.  The  only  way  effectually  to  sterilize  the  cylinder  without 
injuring  it  is  to  place  it  in  a  vessel  with  cold  or  tepid  water,  and  to  boil  it  for  about  an 
hour  "  (Journal  of  Hygiene,  1909,  p.  33).  It  has  already  been  stated  that  simple  boiling 
at  100°  C.,  though  prolonged,  cannot  be  relied  upon  to  destroy  all  micro-organisms.] 


FIG.  15—  Cobbett's  bulb  with  attach- 
ments for  filtration  by  compression. 


FILTRATION   OF   CULTURE   MEDIA 


19 


be  run  off  into  any  suitable  sterile  vessel.     When  the  bulb  is  emptied,  replace  the 
test-tube,  tighten  the  clip,  open  G,  and  repeat  the  operation.] 

2.  The  filtrate  may  also  be  collected  through  a  piece  of  glass  tubing  connected 
by  a  piece  of  india-rubber  tubing  a  few  centimetres  long  to  the  nozzle  of  the  filter 
(fig.  16).  The  bougie,  with  rubber  and  glass  tubing  attached,  is  wrapped  in  paper 


FIG.  16.— Alternative  method 
of  collecting  the  filtrate. 


FIG.   17. — Flask  with  three  tubulures  for  the 
collection  and  distribution  of  the  filtrate. 


and  sterilized.  When  required  for  use,  the  bougie  is  fixed  in  its  metal  cylinder, 
the  paper  removed  from  the  rubber  and  glass  collecting  tubes,  and  the  latter  pushed 
through  the  paper  cap  covering  the  mouth  of  the  sterile  vessel  in  which  the  filtered 
liquid  is  to  be  collected.  If  the  collecting  vessel  be  plugged  with  wool,  the  tube 
is  inserted  between  the  neck  and  the  plug,  the  tube  being  surrounded  as  completely 
as  possible  with  wool  and  pushed  downwards  until  the  orifice  projects  below  the 
wool. 

3.  Another  arrangement  is  to  use  a  flask  with  three  tubulures,  such  for  instance 
as  that  shown  in  fig.  17.  The  flask  must  of  course  be  sterile;  the  wool  in  the  mouth 
of  the  india-rubber  tubing  B  is  removed,  and  the  tube  itself  attached  to  the  nozzle 
of  the  filter.  When  filtration  is  completed  the  tubing  is  removed  from  A,  which 
is  then  plugged  with  a  sterile  plug,  all  necessary  precautions  being  taken  to  prevent 
contamination.  To  manipulate  the  filtrate  the  tubulure  C  is  broken  and  the 
liquid  run  out  by  simply  inclining  the  flask.  The  third  tubulure  D  is  plugged 
with  wool. 

Filtration  by  compression  involves  the  use  of  a  costly  piece  of  apparatus, 
and  is  limited  in  practice  to  the  filtration  of  viscous  fluids. 

B.  Filtration  by  aspiration. 

Aspiration  is  the  means  usually  employed  for  the  filtration  of  fluids.  The 
methods  by  which  the  principle  of  filtering  by  aspiration  is  applied  vary  in 
detail,  and  the  technique  of  a  few  of  the  simplest  and  easiest  devices  will 
be  described. 

[1.  Fit  up  and  sterilize  a  Cobbett's  bulb  exactly  as  described  for  filtration 
under  pressure  (p.  18),  and  clamp  it  to  a  suitable  stand.  Connect  the  bulb 
to  a  wash-bottle  with  a  piece  of  red  rubber  pressure-tubing,  but  between  the 
bulb  and  the  wash-bottle  insert  either  a  three-way  tap  or  a  T-piece  of  glass 
tubing  the  vertical  limb  of  which  is  closed  by  india-rubber  tubing  and  a 
clip,  then  connect  the  wash-bottle  to  the  pump. 


20 


STERILIZATION   BY   FILTRATION 


[Technique.— Stand  the  filter  F  in  a  tall  glass  cylinder  C  which  must  be 
rather  larger  than  the  filter,  and  fill  up  the  cylinder  with  the  unfiltered  liquid. 
Tighten  the  clip  K  on  the  vertical  limb  of  the  T-piece  and  also  the  clip  H  of 
the  delivery  tube,  and  turn  on  the  water.  The  liquid  is  thus  aspirated 


C 


F 


FIG.  18.— Cobbett's  bulb  fitted  up  for  filtration  by  aspiration. 

through  the  filter  into  the  bulb.  When  the  bulb  is  nearly  full,  gently  release 
the  clip  K  on  the  vertical  limb  of  the  T-piece,  and  then  turn  off  the  water. 
Then,  as  before  (p.  18),  remove  the  test-tube  E  and  draw  off  the  filtrate  into 
suitable  and  previously  sterilized  vessels.  Having  emptied  the  bulb  replace 
the  test-tube  E,  tighten  the  clips  K  and  H,  turn  on  the  water  and  exhaust 
again.  In  case  the  filtrate  or  a  part  of  it  has  to  be  stored  for  future  use,  the 
vessels  in  which  it  has  been  collected  may  be  sealed  off  in  the  flame  of  the 
blow-pipe,  or  the  wool  plugs  can  be  made  air-tight  to  prevent  evaporation 
by  sealing  them  with  paraffin  or  sealing-wax  (p.  30).] 

[2.  Instead  of  a  Cobbett's  bulb,  an  Erlenmeyer  flask  may  be  used,  but  the 
procedure  is  a  little  more  complicated  (fig.  19).  The  filtrate  is  aspirated  into 
an  Erlenmeyer  flask,  and  then  blown  out  with  a  bicycle  pump. 

[Technique. — (a)  Take  an  Erlenmeyer  flask  of  sufficient  size  to  contain  the 
filtrate.  Plug  the  lateral  tubulure  with  wool  between  the  constrictions. 


FIG.  19.— A  convenient  arrangement  for  filtration  by  aspiration. 

Fit  the  mouth  with  an  india-rubber  bung  with  two  holes  ;  through  one  hole 
pass  a  piece  of  glass  tubing  bent  at  a  right  angle,  the  vertical  limb  of  which 
is  long  enough  to  reach  to  the  bottom  of  the  flask,  and  through  the  other 


FILTRATION   BY   ASPIRATION 


21 


another  piece  of  glass  tubing  also  bent  at  a  right  angle,  the  vertical  limb  of 
which  reaches  only  just  below  the  level  of  the  lateral  tubulure.  To  the 
horizontal  arm  of  the  latter  attach  a  piece  of  red  rubber  pressure-tubing, 
which  at  its  other  end  is  connected  to  the  nozzle  of  the  filter.  To  the  other 
piece  of  glass  tubing  attach  another  piece  of  pressure-tubing,  into  the  distal 
end  of  which  is  inserted  a  short  piece  of  glass  tubing  drawn  out  to  a  narrow 
orifice  ;  over  this  glass  tube  an  india-rubber  plug  is  slipped  to  fit  a  test-tube 
which  protects  the  end  of  the  delivery  tube. 

[(6)  Sterilize  in  the  autoclave  at  120°  C. 

[(c)  Attach  the  lateral  tubulure  of  the  flask  to  the  lateral  tubulure  of  another 
Erlenmeyer  flask  with  pressure-tubing,  and  insert  a  three-way  piece  of  glass 
tubing  between  the  two  flasks,  the  vertical  limb  being  closed  with  rubber 
tubing  and  a  clip.  Pass  a  right-angled  piece  of  glass  tubing  through  an  india- 
rubber  bung  in  the  mouth  of  the  flask,  and  attach  this  tubing  to  the  pump 
by  means  of  pressure-tubing. 

[(d)  Place  the  filter  in  a  glass  cylinder  larger  than  the  filter,  and  fill  up  the 
cylinder  with  the  fluid  to  be  filtered.  Secure  the  two  clips. 

[(e)  Turn  on  the  pump,  and  the  fluid  is  aspirated  from  the  cylinder  to  the 
Erienmeyer  flask.  When  the  fluid  reaches  nearly  up  to  the  level  of  the 
lateral  tubulure,  release  the  clip  on  the  vertical  limb  of  the  three-way  piece 
of  glass  tubing.  Disconnect  the  second  flask,  and  attach  a  bicycle  pump  to 
the  first  flask.  Clip  the  tubing  attached  to  the  filter. 

[By  working  the  pump  the  flask  can  be  filled  with  air — filtered  by  passing 
through  the  wool  plug  in  the  lateral  tubulure — and  the  contents  of  the  flask 
thus  put  under  sufficient  pressure  to  allow  them  to  be  drawn  off  through  the 
tube  contained  in  the  test-tube,  as  in  the  former  case.] 

3.  A  third  method  is  to  attach  one  end  of  a  piece  of  red  rubber  pressure- 
tubing  B  (fig.  20)  to  the  nozzle  of  a  filter,  and  the  other  end  to  a  piece  of 


FIG.  20.— Filtration  by  aspiration. 


glass  tubing  bent  at  a  right  angle.  Pass  the  latter  through  one  of  the  holes 
in  an  india-rubber  bung.  Fit  the  bung  tightly  into  the  neck  of  a  stout  white- 
glass  bottle  A  the  capacity  of  which  is  equal  to  the  amount  of  fluid  to  be  filtered 
(thin-walled  flasks  will  not  withstand  the  pressure,  and  should  therefore 
never  be  used  for  this  purpose).  Through  the  other  hole  in  the  bung  pass 


22 


STERILIZATION   BY   FILTRATION 


another  piece  of  glass  tubing  also  bent  at  a  right  angle ;  the  vertical  branch 
should  reach  a  few  centimetres  below  the  bung,  while  the  horizontal  arm 

has  two  constrictions  with  a  fairly  tight  plug  of  wool  C  between 

them. 

The  apparatus  thus  fitted  up  is  heated  in  the  autoclave  at 

120°  C.  for  20  minutes.     When  cool,  it  is  examined  to  see  that  the 

bung  still  fits  tightly,  and  the  apparatus  is  then  ready  for  use. 

Technique. — Stand  the  filter  F  in  a  glass  cylinder  E  which  must  be 
rather  larger  than  the  filter,  and  fill  up  the  cylinder  with  the  un- 
filtered  liquid.  Connect  the  horizontal  limb  of  the  tube  D  by 
means  of  pressure-tubing  with  a  water  pump  (Chap.  VI.)  and  exhaust. 
The  liquid  is  thus  aspirated  through  the  filter  into  the  bottle  A. 

When  the  liquid  has  all  passed  through,  turn  off  the  water  and 
disconnect  the  tubing  connecting  the  bottle  and  the  pump  (the  air 
which  will  then  enter  the  bottle  is  filtered  through  the  wool  plug  C 
between  the  constrictions).  Flame  the  neck  of  the  bottle,  and  replace 
the  bung  either  by  a  previously  sterilized  wool  plug  or  by  another 
bung  so  arranged  that  the  fluid  can  be  manipulated  as  described  later. 
The  liquid  thus  sterilized  by  filtration  can  be  kept  sterile  indefinitely 
in  the  bottle. 

There  is  always  a  little  liquid  left  in  the  filter,  and  if  necessary  this 
can  be  collected  by  disconnecting  the  tube  B  from  the  nozzle  and 
aspirating  the  fluid  into  a  long  sterile  bulb  pipette  (fig.  21). 


FIG.  21.-  Bulb 
pipette. 


B 


Distribution  of  the  filtrate. — Having  obtained  a  sterile  filtrate, 
it  follows  that  the  subsequent  manipulations  must  be  so  devised 
as  not  to  contaminate  it.  The  methods  to  be  employed  will  now 
be  described. 

The  bung  used  during  filtration  (p.  21)  must  be  replaced  by  another  fitted 
in  the  following  manner.  Take  an  india-rubber  bung  perforated  with  two 
holes  of  the  same  size  as  the  one  to  be  replaced.  Through  one  hole  pass  a 
piece  of  glass  tubing  A  (fig.  22)  bent  at  a 
right  angle  and  having  a  cotton-wool  plug 
between  constrictions  in  the  horizontal  arm. 
Through  the  other  hole  pass  another  piece 
of  glass  tubing  B  bent  in  the  form  of  an 
inverted  open  U,  one  limb  of  which  should 
reach  nearly  to  the  bottom  of  the  bottle 
while  the  other,  which  will  be  outside  the 
bottle,  is  drawn  out  to  a  fine  capillary  point 
and  sealed .  Wrap  the  bung  with  its  glass  tubes 
in  situ  in  paper,  and  autoclave  at  120°  C. 

Flame  the  neck  of  the  bottle,  remove  the 
paper  covering  from  the  sterilized  bung,  and 
hold  the  latter  by  its  upper  part  in  the  left 
hand.  Take  out  of  the  bottle  with  the  right 
hand  the  bung  used  for  filtration,  and  replace 
it  by  the  other  as  quickly  as  possible  in  order 
to  prevent  dust  falling  into  the  bottle.  Care 
must  of  course  be  taken  that  during  these 
manipulations  the  new  bung  with  its  tubes 
comes  in  contact  with  nothing  likely  to  soil  it. 

To  withdraw  the  fluid,  it  is  only  necessary  to  connect  an  india-rubber 
syringe  to  the  tube  A,  and  after  flaming  the  capillary  end  of  B  to  break  off 
the  point  with  a  pair  of  sterile  forceps.  By  squeezing  the  syringe  a  few 
times  the  liquid  will  be  forced  out  through  B.  When  the  quantity  required 


FIG.  22.— Distribution  of  the  filtrate. 


FILTRATION   BY   ASPIRATION 


23 


has  been  withdrawn  the  end  B  is  sealed  in  a  Bunsen  flame  thus  effectually 
excluding  air  from  the  bottle. 

It  may  however  sometimes  happen  that  on  ceasing  to  work  the  syringe 
some  air  will  enter  the  bottle  through  B,  and  since  this  may 
carry  organisms  with  it  there  is  a  risk  of  the  liquid  in  the  bottle 
becoming    contaminated.     The  difficulty  is  easily  overcome  by 
the  following  simple  device. 

Before  being  sterilized  the  external  limb  of  the  tube  B  is  cut 
in  the  middle,  and  the  two  ends  B  and  C  (fig.  23)  connected  by 
means  of  a  piece  of  red  rubber  tubing,  into  which  a  short  length 
(1-2  cm.)  of  glass  rod  D  has  already  been  introduced.  When 
not  in  use  the  glass  rod  completely  obliterates  the  lumen  of  the 
rubber  tubing,  and  cuts  off  all  communication  between  the 
outside  air  and  the  contents  of  the  bottle.  But  if  the  rubber 
tubing  be  pinched  between  the  thumb  and  index  finger,  a 
small  channel  is  formed  through  which  the  liquid  can  be  forced 
by  squeezing  the  syringe  attached  to  A  (fig.  22).  The  apparatus 
would  then  be  worked  as  follows : 

The  end  of  the  glass  tubing  C  being  flamed  and  the  point 
broken  off,  the  syringe  is  squeezed  a  few  times,  and  then  the 
india-rubber  tubing  between  B  and  C  pinched  up.     The  liquid 
will  then  run  out  from  the  open  end.     The  flow  of  liquid  is   stopper  for  use 
stopped  by  first  releasing  the  finger  and  thumb  from  the  india-   with  distribut- 
rubber    tube,    thus    cutting    off    all   communication   with   the 
outside  air,  and  then  but  not  till  then  relaxing  the  pressure  on  the  syringe. 
Finally,  the   broken   end   is   sealed   and   the   syringe   disconnected. 

4.  L.  Martin's  filtering  apparatus  (fig.  24). — This  consists  of  a  porcelain 


FIG.    23.— 


FIG.  24. -L.  Martin's  filter. 

(The  upper  tubulure  of  the  bulb  should  have  a  wool  plug  between  constrictions : 
this  has  been  accidentally  omitted  from  the  figure.) 

filter  contained  in  a  metal  cylinder  similar  to  that  described  before  (p.  15). 
The  cylinder  has  a  tap  funnel  screwed  into  the  top  to  facilitate  manipulation. 
Technique. — Connect  the  nozzle  of  a  porcelain  filter  with  a  bulb  of  the  shape 
shown  in  the  figure  by  a  length  of  pressure-tubing.  Sterilize  in  the  autoclave. 
Then  fix  the  filter  in  its  metal  case,  and  connect  the  upper  tubulure  of  the  bulb 
to  a  water  pump  with  pressure-tubing. 


24 


STERILIZATION   BY   FILTRATION 


Fill  the  cylinder  through  the  funnel  with  the  fluid  to  be  filtered,  close  the  tap 
and  turn  on  the  water.  The  fluid  in  the  cylinder  is  aspirated  through  the  filter, 
along  the  tubing,  and  so  into  the  bulb.  When  all  the  liquid  has  been  aspirated, 
turn  off  the  water,  open  the  tap,  and  disconnect  the  bulb  from  the  water  pump.  The 
lower  tubulure  of  the  bulb  is  sealed  during  filtration,  but  is  flamed  and  the  point 
broken  off  with  sterile  forceps  before  distributing  the  filtrate. 

This  is  a  useful  piece  of  apparatus,  but  its  cost  is  a  disadvantage. 

5.  Chamberland's  method. — If  water  be  not  available,  a  small  hand  pump, 
e.y.  Potain's,  may  be  used  for  aspiration.  Place  the  filter  B  (fig.  25)  in  a 


FIG.  25.— Chamberland's  filter. 

tall  glass  cylinder  C,  and  fill  up  the  latter  with  the  liquid  to  be  filtered.  On 
working  the  aspirator  the  fluid  is  drawn  through  the  filter  into  the  flask  A, 
which  has  three  tubulures.  The  filter  and  flask  must  both  be  sterilized  in 
the  autoclave  before  use. 

[It  will  be  obvious,  of  course,  that  Cobbett's  bulb  can  be  used  equally  with 
a  hand  or  water  pump.] 

3.  The  filtration  of  small  quantities  of  liquid. 

The  laboratory  bougie. — When  only  very  small  quantities  of  liquid  have 
to  be  filtered,  as  for  example  in  testing  a  toxin,  a  small  thin-walled  bougie 

12-15   cm.    long   and   without   a 
nozzle  is  very  useful. 

Technique. — [A.  Slip  one  end  of  a 
piece  of  stout  pressure- tubing  over 
the  open  end  of  the  bougie  and  secure 
it  with  a  rubber  ligature,  then  con- 
nect the  other  end  to  the  free  limb 
of  the  U-tube  of  a  Cobbett's  bulb. 
Sterilize  in  the  autoclave.  Place  the 
filter  in  a  small  glass  cylinder  or  test- 
tube  and  fill  the  latter  with  the  fluid 
to  be  filtered.  Connect  the  bulb  to  a 
water  pump  and  exhaust.  Any  fluid 
remaining  in  the  filter  can  be  recov- 
ered by  holding  the  filter  upside  down, 
and  allowing  it  to  run  into  the  bulb.] 
B.  The  filter  may  also  be  arranged  as  shown  in  fig.  26.  As  in  A,  a  piece  of  pressure- 
tubing  is  firmly  fixed  by  one  end  to  the  upper  end  of  the  filter,  but  the  other  end  is 
attached  to  one  of  the  two  tubulures  of  the  flask  B.  The  other  tubulure  A  is  plugged 
with  cotton- wool  and  connected  to  a  water  pump  or  a  small  aspirator,  e.g.  Potain's. 
Sterilize  the  apparatus  before  use. 


FIG.  26.— Laboratory  bougie  for  filtering  small 
quantities  of  liquid. 


FILTRATION   OF   SMALL   QUANTITIES   OF   FLUID         25 

C.  Duclaux's  filter. — The  filter  can  also  be  fitted  to  a  flask  with  three  tubulures 
(fig.  27).  In  this  case  the  open  end  of  the  filter  is  wrapped  round  with  cotton- wool, 
which  serves  to  hold  the  bougie  in  position  in  the  neck  of  the  upper  tubulure  E. 
The  tubulure  D  is  sealed,  and  B  is  plugged  with  wool.  After  autoclaving,  the  wool 
packing  around  the  neck  of  the  filter  F  is  made  air-tight  by  running  a  little  melted 


FIG.  27. — Laboratory  bougie — Duclaux's 
arrangement. 


FIG.  28. — Laboratory  bougie — Kitasato's 
arrangement. 


wax  ([paraffin  or]  Golaz's)  over  it.  The  tubulure  B  is  connected  to  a  water  pump, 
and  the  liquid  to  be  filtered  poured  into  E.  On  turning  on  the  water  the  liquid 
is  drawn  through  the  filter  and  collects  in  the  flask.  To  distribute  the  filtrate, 
break  off  the  sealed  end  D  and  blow  air  into  the  flask  through  the  wool-plugged 
orifice  B. 

D.  Kitasato's  filter  (fig.  28)  consists  of  a  conical  flask  of  thick 
glass  furnished  with  a  lateral  tube  b,  which  when  in  use  is  plugged 
with  wool  and  connected  to  a  water  pump. 

The  wide  neck  of  the  flask  is  fitted  with  a  perforated  india- 
rubber  bung  through  which  the  filter  F  is  passed.  The  mouth 
of  the  filter  is  attached  by  means  of  another  india-rubber  bung  to 
a  glass  bulb  A.  The  technique  is  very  simple :  pour  the  liquid 
into  A,  turn  on  the  water  pump,  and  the  filtrate  collects  in  the 
flask  B.  The  apparatus  must  of  course  be  autoclaved  before  use. 

E.  Martin's  filter  (fig.  29),  as  arranged  for  dealing  with  small 
quantities  of  fluid,  consists  of  a  tube  R,  which  can  be  connected 
to   a  water   pump   through   the  tubulure  A.     Within   it   is   a 
moderately  large  test-tube  T  resting  upon  a  pad  of  cotton- wool. 
A  small  filter  F  is  passed  into  the  test-tube,  and  firmly  fixed  in 
the  mouth  with  the  open  end  upwards.     The  tube  R  is  closed 
above  with  an  india-rubber  bung,  through  which  passes  a  glass 
funnel  E  the  lower  end  of  which  is  connected  with  the  upper 
(open)    end   of   the   filter.     The  liquid  to  be  filtered  is  poured 
into  E,  and  being  drawn  through  the  filter  collects  in  the  tube  T. 

FIG.   29. 

To  sum  up,  there  are  three  important  considerations  to  be   arrangement.1' 
kept  in  mind  when  using  a  filter  for  purposes  of  sterilization. 

(I)  In  every  case  the  filter  must  be  tested  to  make  sure  it  is  sound  and  free 
from  fissures ;  (2)  filters  must  always  be  sterilized  immediately  before  use ; 
and  (3)  subsequent  contamination  of  the  filtrate  must  be  carefully  guarded 
against. 


26  STERILIZATION   BY  ANTISEPTICS 


SECTION  IV.— STERILIZATION   BY   ANTISEPTICS. 

Sterilization  by  antiseptics  has  but  limited  use  in  bacteriological  work. 
The  addition  of  antiseptics  will  it  is  true  destroy  micro-organisms  in  a 
medium  designed  for  the  growth  of  cultures,  but  the  amount  of  antiseptic 
which  has  to  be  added  to  effect  this  result  is  very  much  greater  than 
the  amount  required  to  inhibit  the  multiplication  of  any  organisms 
which  may  subsequently  be  sown  in  it ;  the  medium  is  therefore  rendered 
useless. 

1.  Antiseptics  are,  however,  in  general  use  for  sterilizing  the  interior  of 
glass  dishes,  bell  jars,  and  other  similar  articles  which  are  to  be  used  to 
protect  from  dust  and  contamination  Petri  dishes,  culture  tubes,  etc.,  and 
which  will  not  come  in  contact  with  any  culture  medium,  or  with  the  organisms 
under    investigation.      Fixed    non-volatile    antiseptics    must   be    employed 
since  the  vapours  given  off  by  volatile  compounds  hinder  the  growth  of 
organisms  on  culture  media. 

A  0*1  per  cent,  solution  of  perchloride  of  mercury  may  be  used.  The 
solution  should  be  made  with  distilled  water,  but  if  tap  water  be  used  a 
small  amount  (O5-1  gram)  of  tartaric,  acetic  or  hydrochloric  acid  must 
be  added  to  prevent  precipitation  of  the  mercury  salt  by  the  salts  dissolved 
in  the  water. 

Perchloride  of  mercury  has  however  now  been  almost  entirely  discarded 
in  favour  of  oxycyanide  of  mercury  in  Ol  per  cent,  solution.  This  solution 
though  powerfully  antiseptic  has  no  caustic  action,  it  does  not  precipitate 
albuminoid  substances,  neither  does  it  attack  instruments  and  other  metal 
articles. 

2.  Antiseptics  are  also  in  general  use  for  sterilizing  the  hands,  and  for 
washing    out   vessels    and    sterilizing    instruments   during   inoculation    and 
other   experiments.      Solutions    of   Ol    per   cent,    of    perchloride    or    oxy- 
cyanide of  mercury  or  1*5  per  cent,  of  formalin  are  often  used   for  these 
purposes. 

[Lysol,  a  solution  of  the  three  cresols  in  soap  and  water,  is  a  particularly 
useful  antiseptic.  In  2  per  cent,  solution  it  does  not  hurt  the  skin,  and  the 
soap  in  solution  makes  a  lather  if  the  hands  be  washed  in  it  or  if  the  surface 
of  the  skin  be  rubbed  with  a  sponge  soaked  in  the  solution ;  the  presence 
of  the  soap  makes  a  solution  of  lysol  a  more  efficient  antiseptic  for  these 
purposes  than  mercury  solutions.  Lysol  does  not  damage  metal  instruments, 
and  does  not  precipitate  albuminoid  solutions.  If  made  up  in  large  quan- 
tities with  hard  water  the  soap  is  liable  to  be  precipitated  to  some  extent, 
but  the  antiseptic  constituents  still  remain  in  solution.] 

Solutions  of  perchloride  or  oxycyanide  of  mercury  may  also  be  used  for 
sterilizing  the  surface  of  the  skin  before  collecting  pus,  blood,  etc.,  from  the 
living  subject  (Chap.  XII.).  Care  must  of  course  be  taken  that,  after  steriliza- 
tion, all  traces  of  the  antiseptic  are  removed  by  washing  the  part  well  with 
alcohol  before  collecting  the  material,  otherwise  the  presence  of  the  anti- 
septic would  materially  interfere  with  the  subsequent  growth  of  organisms 
in  culture.  [At  the  present  time,  however,  it  is  more  usual  to  paint  the  sur- 
face of  the  skin  with  tincture  of  iodine  (British  Pharmacopoeia)  before 
penetrating  it  for  the  purpose  of  collecting  material  for  bacteriological 
investigation.] 

3.  Antiseptics   are   also   added   to    sterile   filtrates   which  are   no   longer 
required  as  culture   media.     For  this  purpose  a   small   quantity  of  some 
antiseptic  (such  as  thymol  or  camphor)  which  is  without  chemical  action 
on  the  constituents  of  the  fluid  is  selected. 


STERILIZATION   BY   ANTISEPTICS  27 

[Wright  adds  a  trace  (0*5  per  cent.)  of  carbolic  acid  to  his  vaccines.] 
4.  Antiseptics  are  sometimes  used  to  sterilize  a  culture  when  the  products 
of  micro-organisms  are  under  investigation.  Volatile  antiseptics  such  as 
chloroform,  ether,  toluol,  essence  of  garlic  or  mustard,  etc.,  which  can  be 
readily  driven  off  afterwards  by  evaporation,  are  the  most  useful  in  this 
connexion. 


CHAPTER  II. 
CULTURE  MEDIA. 

Introduction. 

Section  I. — Liquid  media,  p.  30. 

1.  Media  made  from  animal  tissues  and  fluids,  p.  30.     2.  Media  made  from  vegetable 
tissues,  p.  37.     3.  Synthetic  media,  p.  38. 
Section  II. — Solid  media,  p.  39. 

1.  Gelatin  media,  p.  39.  2.  Agar  media,  p.  42.  3.  Media  made  from  albuminous 
fluids  and  tissues, — serum,  egg,  etc.,  p.  45.  4.  Media  made  from  vegetable  tissue, 
p.  55.  5.  Coloured  media,  p.  56. 

THE  substances  requisite  for  the  growth  of  micro-organisms  may  be  obtained 
by  macerating,  infusing  or  boiling  tissues  of  animal  or  vegetable  origin. 
Saline  solutions  in  which  some  carbo-hydrate  is  dissolved  also  supply  all  the 
ingredients  essential  for  a  culture  medium. 

Culture  media  are  either  solid  or  liquid. 

Chemically,  like  all  other  living  cells,  micro-organisms  consist  of  organic  and 
inorganic  nitrogen  and  mineral  salts  ;  it  is  therefore  necessary  in  order  to  grow  a 
micro-organism  that  these  three  classes  of  substances  be  made  available,  together 
with  oxygen  which  is  an  essential  to  the  life  of  all  living  structures.  [Finally,  a 
certain  amount  of  moisture  is  absolutely  necessary.] 

Micro-organisms  are  divided  into  two  large  groups,  the  members  of  one  of  which 
derive  their  oxygen,  like  more  highly  organized  structures,  from  the  free  oxygen 
of  the  atmosphere,  while  the  members  of  the  other  group  cannot  multiply  in  presence 
of  free  oxygen,  but  obtain  the  oxygen  they  require  by  the  decomposition  of  sub- 
stances containing  it  (Pasteur).  The  former  are  known  as  the  aerobic,  the  latter 
as  the  anaerobic  organisms. 

These  two  groups  of  micro-organisms  call  for  very  different  methods  of  artificial 
cultivation.  Aerobic  micro-organisms  should  be  grown  in  vessels  in  which  there 
is  an  ample  supply  of  air  ;  anaerobic  micro-organisms  on  the  other  hand  only 
grow  if  air  be  excluded.  The  latter  therefore  are  cultivated  either  in  vacuo,  or 
in  presence  of  some  inert  gas. 

The  constituents  of  culture  media  are  however  the  same  for  both  aerobic  and 
anaerobic  organisms  and  ought  to  include  nitrogen  compounds  and  salts  of  the 
ternary  bases.  Many  organisms  can  convert  inorganic  nitrogen  (nitrates,  etc.) 
into  organic  nitrogen,  while  in  some  cases  organisms  will  grow  in  purely  inorganic 
solutions  provided  these  contain  a  small  quantity  of  some  carbohydrate  such 
as  sugar. 

General  Rules. — Every  culture  medium  therefore  must 

(1)  contain  the  substances  necessary  for  the  growth  of  the  organism  to  be  sown 
[(2)  be  of  suitable  reaction] ;  (3)  have  been  previously  sterilized  ;  (4)  be  con- 
tained in  vessels  which  afford  protection  from  contamination  from  without. 


CULTURE   VESSELS 


29 


Culture  vessels. 

Vessels  of  various  patterns  are  used  for  culture  media,  and  these  will  be 
described  as  occasion  for  their  use  arises.  In  this  chapter  a  description  of 
those  most  commonly  used  for  the  growth  of  aerobes  only  will  be  given. 

1.  Ordinary  test-tubes,  but  without  lips,  are  in  constant  use  (fig.  30)  ;  they 
must  be  plugged  with  wool  as  already  described. 

2.  Erlenmeyer  flasks— conical  glass  vessels  with  a  flat  bottom  (fig.  31)  and 
of  different  sizes— [and  Jena  flasks,]  are  also  in  frequent  use.     Ordinary  small 


&>3 


FIG.  30. — Culture  tube 
plugged  with  wool. 


FIG.  31. — Erlenmeyer  flask. 


medicine  bottles  of  30  to  50  c.c.  capacity  can  be  used  in  many  cases.  What- 
ever be  the  shape  of  the  vessel  it  must  be  plugged  with  wool,  and  as  a  further 
protection  a  paper  cap  is  useful  (vide  p.  4). 

3.  Small  vessels  capable  of  holding  30  to  50  grams  and  known  as  Pasteur's  flasks,  are 
also  in  frequent  use  [in  France].  The  mouths  of  these  flasks  are  generally  closed  by 
means  of  an  hollow  ground-glass  stopper  fitting  a  similarly  ground-glass  surface  on  the 
neck  of  the  flask,  and  having  a  small  orifice  above,  which  must  be  plugged  with  wool. 
This  method  of  plugging  effectually  preserves  the  contents  of  the  flasks  from  contamina- 
tion, but  has  the  disadvantage  of  being  very  fragile,  and  the  glass  cap  is  often  broken  in 
the  flaming  process  preliminary  to  opening  the  flask. 

It  is  better  to  cover  the  mouth  of  the  flask  with  a  small  paper  hood  (this  can  be  done 
by  enveloping  the  neck  in  a  small  strip  of  filter  paper  and  tightly  screwing  the  projecting 
part  into  a  point).  It  is  even  simpler  to  plug  the  flasks  with  wool  in  the  same  way  as 
test-tubes  are  plugged. 

Miquel's  flask  is  merely  a  conical  form  of  Pasteur's  pattern. 

Prevention  of  evaporation. 

Evaporation  readily  takes  place  through  a  wool  plug,  and  if  a  medium — especially 
a  solid  medium  such  as  potato,  serum,  agar,  etc. — be  stored  or  incubated  for  a 
long  time,  the  amount  of  evaporation  is  likely  to  be  excessive.  To  avoid  this,  the 
mouth  of  the  vessel  may  be  closed  with  an  india-rubber  cap.  This  must  be 
sterilized  before  use,  because  when  the  cap  is  slipped  over  the  mouth  of  the  tube  or 
flask  the  air  within  being  saturated  with  aqueous  vapour  will  soon  make  the  wool 
plug  moist,  and  then  any  organism  on  the  inner  surface  of  the  cap  will  ultimately 
grow  through  the  moist  plug  and  contaminate  the  contents. 


30  LIQUID   MEDIA 

Red  rubber  caps  are  the  best ;  they  should  be  put  into  a  wide- mouthed  flask 
or  bottle,  which  is  then  plugged  with  wool  and  autoclaved  and  afterwards  put  in 
the  incubator  to  dry  the  plug.  A  cap  can  then  be  taken  out  with  a  pair  of  sterile 
forceps  whenever  one  is  wanted. 

In  the  case  of  stock  cultures,  which  are  to  be  put  away  for  some  time, 
evaporation  maybe  prevented  by  pouring  a  little  melted  paraffin  [or  sealing- 
wax]  over  the  top  of  the  plug. 

[This  latter  method  of  sealing  tubes  or  bottles  is  also  of  great  use  in  the 
cultivation  of  slow-growing  organisms  such  as  the  tubercle  bacillus.  After 
sowing  the  medium,  the  top  of  the  plug  is  carefully  sealed  with  melted 
paraffin  (or  sealing-wax),  and  the  culture  can  then  be  incubated  as  long  as 
is  necessary  without  fear  of  the  medium  drying  up,  if  the  sealing  has  been 
efficiently  done.] 

Method  recommended. — [When  paraffin  is  used,  gently  warm  the  upper 
J  cm.  of  the  tube  by  turning  it  round  in  the  flame,  and  then  with  a  pipette 
or  ladle  pour  a  few  drops  of  melted  paraffin,  kept  liquid  in  a  water  bath,  on 
to  the  warm  wool  and  let  it  soak  in  to  a  depth  of  |  cm.  or  so.  To  unseal 
the  plug,  gently  warm  the  upper  part  of  the  tube  again,  stick  a  needle  or 
pair  of  forceps  into  the  plug  and  turn  it  round  at  the  same  time  raising  it. 

[Another  simple  method  of  preventing  evaporation  during  cultivation  in  test- 
tubes  is  to  place  them  in  a  large  wide-mouthed  ground-glass  stoppered  jar,  which 
has  been  previously  thoroughly  washed  out  with  a  saturated  solution  of  perchloride 
of  mercury.  Place  two  or  three  folds  of  filter  paper  moistened  with  perchloride 
solution  at  the  bottom  of  the  bottle,  and  after  arranging  the  tubes  put  a  trace  of 
vaseline  on  the  stopper  and  close  the  bottle,  turning  the  stopper  round  to  obliterate 
any  air  channels.  Volatile  antiseptics  (e.g.  formalin)  are  obviously  unsuitable  for 
this  purpose.] 

SECTION  I.— LIQUID   MEDIA.1 
1.  Media  made  from  animal  tissues  and  fluids. 

There  is  a  great  variety  of  these  media.  A  description  of  those  in  most 

general  use  only  will  be  given  here  ;    and  the  most  frequently  used  of  all, 

peptone  beef  broth,  will  be    taken   as   a  type    and  the   technique    of   its 
preparation  given  in  fullest  detail. 

Peptone  beef  broth. 

This  medium  is  in  everyday  use.  It  will  be  referred  to  in  future  simply 
as  broth. 

Preparation. — 1.  Take  500  grams  of  lean  beef.  Cut  away  all  fat,  tendon 
and  aponeurosis.  Mince  it,  and  leave  it  to  macerate  in  a  litre  of  cold  water 
for  6  to  12  hours. 

2.  Heat  gently  to  boiling  in  an  enamelled  saucepan,  stirring  constantly, 
and  keep  the  mixture  boiling  for  10  minutes. 

3.  Pour  on  to  a  thick  clean  cloth,  express  as  far  as  possible  all  the  fluid 
out  of  the  meat,  and  while  still  warm  filter  the  fluid  through  a  thick  filter 
paper  (Chardin  or  Prat-Dumas)  moistened  with  water  to  keep  back  the  fat. 

4.  Pour  the  filtered  broth  into  an  enamelled  saucepan,  and  add 

Dry  peptone  (Chapoteaut)         -  -         10  grams,  or  1  per  cent,  of  the 

volume  of  water  used. 

Salt,       -  5  grams,  or  0*5  per  cent. 

Sodium  phosphate,  -         -         about  1  gram. 

Boil  again,  stirring  meanwhile  to  dissolve  the  peptone 

1  The  present  chapter  will  be  limited  to  a  description  of  the  culture  media  of  general 
application.  Media  applicable  only  to  particular  organisms  will  be  dealt  with  when  the 
latter  are  under  consideration. 


ANIMAL   TISSUES   AND   FLUIDS  31 

The  addition  of  the  sodium  phosphate  is  not  absolutely  necessary.  Cache  states 
that  the  addition  of  magnesium  salts  increases  the  value  of  the  culture  medium, 
and  advises  the  addition  of  2  grams  of  magnesium  phosphate  per  litre  to  ordinary 
broth,  in  place  of  the  sodium  phosphate.  Magnesium  phosphate  should  be  added 
while  the  meat  is  macerating  (Stage  1  above). 

5.  The  liquid  is  now  strongly  acid  and  must  be  neutralized,  since  bacteria 
grow  best  in  a  neutral  or  slightly  alkaline  medium. 

Neutralization. — To  neutralize  the  medium,  add  normal  soda  solution  to 
the  broth  in  small  quantities  at  a  time  with  a  pipette,  testing  the  reaction 
at  frequent  intervals  against  litmus  paper.  When  a  drop  of  the  broth  placed 
on  a  red  litmus  paper  with  the  end  of  the  stirring  rod  turns  it  slightly  blue, 
sufficient  soda  has  been  added.  The  reaction  should  be  very  slightly  alkaline 
to  litmus,  but  acid  to  phenol-phthalein. 

Neutralization  is  the  most  difficult  step  in  the  preparation  of  broth.  The  amount 
of  alkali  to  be  added  varies  considerably  with  different  pieces  of  meat,  and  can 
only  be  determined  by  trial.  Add  the  soda  solution  very  slowly  stirring  carefully 
after  each  addition ;  and  as  the  neutral  point  is  approached,  test  the  broth  after 
the  addition  of  each  drop  of  alkali  against  both  a  red  and  a  blue  paper.  A  point 
is  ultimately  reached  when  a  drop  of  the  liquid  produces  no  change  on  either  paper; 
it  is  then  sufficient  to  add  a  very  small  quantity  of  soda  solution  to  attain  the 
requisite  degree  of  alkalinity.  According  to  Park  and  Williams,  7  c.c.  per  litre  of 
normal  soda  should  be  added  to  a  neutral  broth  to  obtain  the  most  favourable 
medium.1 

[Eyre  uses  phenol-phthalein  as  the  indicator  and  standardizes  after  Stage  3 
before  the  addition  of  peptone  and  salt. 

[Technique. — 1.  Heat  the  merft  extract  in  the  steamer  at  100°  C.  for  45  minutes. 

2.  Measure  25  c.c.  into  a  beaker  and  add  about  0*5  c.c.  of  a  0-5  per  cent,  solution  of 
phenol-phthalein  in  50  per  cent,  alcohol. 

3.  Immerse  the  beaker  in  a  water  bath  and  raise  to  boiling  point. 

4.  Neutralize  at  the  boiling  point  with  deci-normal  NaOH  solution. 

[The  reaction  is  expressed  by  stating  the  number  of  cubic  centimetres  of  normal 
alkali  required  to  render  one  litre  of  the  meat  extract  exactly  neutral  to  phenol- 
phthalein. 

[For  the  majority  of  organisms  a  medium  which  requires  the  addition  of  10  c.c. 
of  normal  alkali  per  litre  of  meat  extract  is  found  to  be  the  best.  In  Eyre's  scale 
the  reaction  of  such  a  medium  is  expressed  as  -f- 10 :  +  indicating  that  the  medium 
is  acid  and  10  that  it  is  acid  to  the  extent  of  10  c.c.  of  normal  alkali  per  litre.] 

6.  Now  pour  the  slightly  alkaline  broth  into  a  glass  flask,  or  better  into 
an  enamelled  vessel,  and  autoclave  at   115°-117°  C.   for   5    minutes.     The 
liquid  becomes  cloudy  and  deposits  crystals  of  earthy  phosphates. 

On  taking  out  of  the  autoclave,  filter  while  hot  through  a  Chardin  paper 
moistened  with  water :  the  filtrate  should  be  absolutely  clear.  The  object 
of  this  procedure  is  to  remove  any  excess  of  earthy  phosphates,  and  if  omitted, 
the  broth  is  likely  to  become  cloudy  when  sterilized. 

7.  Add  sufficient  distilled  water  to  the  filtrate  to  make  the  total  volume 
up  to  1  litre. 

8.  This  completes  the  preparation  of  the  broth,  which  has  now  only  to 
be  distributed  into  suitable  vessels  and  sterilized. 

Sterilization. — (A)  If  the  broth  is  to  be  kept  for  future  use,  it  may  be 
sterilized  in  a  large  flask  the  neck  of  which  is  either  plugged  with  wool  or 
drawn  out  in  the  flame  and  sealed.  The  medium  can  thus  be  kept  indefinitely, 
and  when  required  for  use  can  be  distributed  into  suitable  vessels. 

(B)  It  is  however  usually  more  convenient  to  distribute  the  broth  at  once 
into  test-tubes  or  small  flasks. 

1  Normal  soda  contains  40  grams  of  NaOH  per  litre  of  distilled  water. 


32  LIQUID   MEDIA 

1.  Into  each  test-tube  put  10-15  c.c.  of  broth,  and  into  each  flask  25  c.c. 
A  small  glass  funnel  should  be  utilized  for  distributing  the  broth,  because 

if  a  drop  of  it  come  in  contact  with  the  wool  plug  it  will  when  dry  cause  the 
wool  to  stick  to  the  mouth  of  the  tube,  so  that  the  plug  can  only  be  removed 
with  difficulty.  (The  use  of  a  funnel  is  even  more  important  when  solid 
media,  such  as  agar  or  gelatin,  are  being  tubed.) 

2.  Plug  the  tubes  and  flasks  with  wool. 

3.  Place  the  vessels  containing  the  medium  in  a  wire  basket,  and  auto- 
clave for  20  minutes  at  110°-115°  C.,  taking  care  that  the  latter  tempera- 
ture is  not  exceeded,  and  that  the  precautions  noted  in  Chapter  I.    are 
observed.1  2 

Veal  broth. 

For  the  preparation  of  veal  broth  proceed  as  for  beef  broth,  using  instead 
of  beef  500  grams  of  lean  veal. 

Chicken  broth. 

Chicken  broth  is  prepared  in  a  similar  manner,  using  500  grams  of  chicken 
meat.  All  skin,  tendon,  and  bone  must  be  removed,  otherwise  the  broth 
will  be  of  a  gelatinous  consistency. 

Giblet  broth. 

Liver,  spleen,  etc.,  may  be  used  for  making  broth.  The  technique  is  the 
same  as  in  the  preceding  cases,  substituting  500  grams  of  the  solid  organs 
for  beef  or  other  meat.  Very  often  these  broths  exhibit  a  slight  cloudiness, 
but  this  cannot  be  avoided. 

Meat  extract. 

1.  To  500  grams  of  well-minced  lean  beef  or  veal,  add  1000  grams  of  water, 
and  leave  in  the  ice  chest  to  macerate  for  12  hours. 

2.  Shake  the  mixture,  filter  through  a  cloth  and  squeeze  all  the  fluid  out 
of  the  meat,  then  filter  through  a  Chardin  paper. 

3.  Add  5  grams  of  salt  to  the  filtrate,  and  heat  to  boiling, 

4.  Neutralize  as  in  the  case  of  peptone  beef  broth. 

5.  Autoclave  for  5  minutes  at  115°— 117°  C. 

6.  While  warm,  filter  through  a  moistened  Chardin  filter  paper. 

7.  Add  sufficient  distilled  water  to  make  up  the  volume  to  a  litre. 

8.  Distribute  in  tubes  and  sterilize  at  110°-115°  C. 

Martin's  peptone  solution. 

Pigs'  stomach  broth. 

1.  Wash,  clean  and  mince  finely  4  or  5  pigs'  stomachs. 

It  is  better  to  use  a  number  (5)  of  stomachs  in  order  to  neutralize  variations  in 
their  pepsin  content ;  if  this  be  done,  the  broths  will  have  an  almost  constant 
average  composition. 

2.  Mix  the  following  : 

Minced  stomachs,    -         -  -         200  grams. 

Hydrochloric  acid  (pure),  .....  10       ,, 

Water  at  50°  C.,       -  -       1000       „ 

and  keep  the  mixture  at  a  temperature  of  50°  C.  for  20-24  hours. 

1  Sometimes  it  will  be  noticed  that  the  medium  when  taken  out  of  the  autoclave  is 
very  slightly  cloud}7 ;  this  will  vanish  on  cooling.  But  if  during  sterilization  the  tem- 
perature exceed  the  temperature  of  the  first  autoclaving  (par.  6,  above),  the  broth  remains 
permanently  cloudy. 

[ 2  In  England  it  is  more  usual  to  sterilize  media  by  steaming  for  20  minutes  on  each 
of  three  successive  days  in  a  Koch's  steamer,  or  some  suitable  modification  of  it  (Chap.  I.).} 


ANIMAL   TISSUES   AND   FLUIDS  33 

3.  Then  heat  to  boiling  to  destroy  the  excess  of  pepsin,  and  pass  through  a 
sieve  or  a  thin  layer  of  loosely  packed  absorbent  cotton- wool. 

4.  Heat  the  filtrate  to  80°  C.,  and  neutralize  at  this  temperature  :    large 
flocculent  masses  are  precipitated ;  filter  the  clear  supernatant  fluid  through 
a  Chardin  paper. 

5.  Autoclave   the   nitrate   for   five   minutes  at  117°-120°  C.,  and  again 
filter  through  a  Chardin  paper. 

6.  Distribute  the  clear  filtrate  in  tubes,  and  sterilize  for  15  to  20  minutes 
at  115°  C. 

Martin's  peptone  broth. 

1.  Mince  500  grams  of  lean  veal,  and  macerate  in  1000  grams  of  water 
for  15  to  20  hours  at  a  temperature  of  35°  C.  to  get  rid  of  the  sugars. 

2.  Filter  through  a  cloth,  squeeze  out  as  much  of  the  fluid  as  possible,  and 
add  5  grams  of  salt. 

3.  Mix   this   liquid   with   an   equal   volume   of  Martin's  peptone  solution 
(see  above,  Stages  1,  2,  3,  4). 

4.  Heat  to   70°  C.   to   coagulate  the  albuminoid  compounds,   and  make 
exactly  neutral.     Then  add  7  c.c.  of  normal  soda  solution  per  litre.     Filter 
through  a  Chardin  paper. 

5.  Sterilize  by  filtering  through  a  Chamberland  bougie  (Chap.   I.),  and 
distribute  in  sterile  culture  vessels. 

Note. — A  broth  prepared  in  this  way  is  particularly  useful  for  the  preparation 
of  diphtheria  toxin.1  For  everyday  use,  it  is  simpler  to  sterilize  by  heat.  After 
.Stage  4  proceed  thus  : 

5a.  Pour  the  broth  into  an  enamelled  vessel  or  flask,  autoclave  for  5  minutes 
at  115°-117°  C.,  and  filter  in  the  warm  through  a  Chardin  paper. 

5b.  Distribute  the  filtered  liquid  into  culture  vessels  and  sterilize  for  20  minutes 
at  110°-115°C.  The  broth  is  often  slightly  and  permanently  cloudy. 

Koch's  peptone  solution. 

1.  Dissolve  in  the  warm,  10  grams  of  peptone  (Witte  or  Chapoteaut)  and 
5  grams  of  salt  in  1000  grams  of  water. 

It  is  unnecessary  to  neutralize  :    peptone  itself  is  sufficiently  alkaline. 

2.  Boil.     Filter." 

3.  Distribute  in  tubes  or  flasks.     Sterilize  at  115°  C. 

Metchnikoff's  peptone-gelatin  medium. 

1.  Dissolve  in  the  warm,  in  1000  grams  of  water, 

Peptone  (Chapoteaut),      -  10  grams. 

Salt,       -  5 

Gelatin  (extra  quality  white),  -  20         „ 

2.  Make  very  slightly  alkaline  with  normal  soda  solution. 

3.  Autoclave  for  5  minutes  at  115°  C.     Filter  through  Chardin  paper. 

4.  Distribute  in  suitable  vessels.     Sterilize  at  HO0-^0  C. 

Miquel's  peptone  solution. 

1.  Dissolve  at  a  moderate  heat  in  a  litre  of  water, 

Peptone  (Chapoteaut),      -          -  20  grams. 

Salt,       -  5     » 

2.  Add  O'lO  gram  of  wood  ash.     Boil.     Filter  through  Chardin  paper. 

3.  The  liquid  is  now  generally  markedly  alkaline.     Neutralize  very  care- 
fully with  st»  solution  of  tartaric  acid,  watching  the  reaction  meanwhile  by 
testing  against  litmus  paper. 

1  Numerous  formulse  for  the  preparation  of  broth  suitable  for  the  study  of  diphtheria 
toxin  have  been  published  (Chap.  XV.). 

C 


34  LIQUID  MEDIA 

4.  Boil  for  5  minutes.     Filter.     Add  sufficient  water  to  make  the  volume 
up  to  a  litre. 

5.  Distribute  in  tubes.     Sterilize  at  115°  C. 

Liebig's  broth. 

1.  Dissolve  5  grams  of  Liebig's  extract  of  meat  ["  Lemco  "]  in  1000  grams 
of  water,  warming  gently.     Neutralize  if  necessary. 

2.  Autoclave  for  5  minutes  at  115°-117°  C.     Filter  through  a  moistened 
paper  in  the  warm. 

3.  Distribute  in  tubes.     Sterilize  at  110°-115°  C. 

Peptone  (Chapoteaut)  (10  grams)  and  salt  (5  grams)  may  be  added  to  the  medium 
before  neutralization. 

In  the  same  way  a  nutrient  broth  may  be  prepared  with  Cibils'  extract. 
In  that  case  20  grams  of  the  extract  is  used  instead  of  the  Liebig. 

These  media  are  used  chiefly  in  German  laboratories,  [and  the  former  to  a  large 
extent  also  in  England]. 

Thymus  broth  (Brieger). 

1.  Obtain  the  thymus  from  two  or  three  calves  directly  after  they  are 
slaughtered.    Mince  the  glands  finely  and  add  an  equal  weight  of  distilled  water. 

Mix,  and  macerate  for  12  hours. 

2.  Filter  through  muslin  and  squeeze  out  as  much  of  the  fluid  as  possible. 
Add  an  equal  volume  of  water  to  the  cloudy  viscous  filtrate. 

3.  Make  feebly  alkaline  with  a  10  per  cent,  solution  of  sodium  carbonate. 

4.  Heat  to  100°  C.  for  15  minutes  in  the  autoclave  or  steamer  (a  higher 
temperature  interferes  with  the  properties  of  the  medium). 

Filter  through  a  piece  of  fine  linen. 

5.  Distribute  in  sterile  tubes. 

Sterilize  at  100°  C.  for  15  minutes  on  each  of  two  successive  days. 

Some  micro-organisms,  such  as  the  cholera  vibrio,  will  only  grow  satisfactorily 
on  this  medium  provided  that  5  or  6  times  its  volume  of  sterile  water  be  added 
just  before  use. 

Serum  broth.    Blood  broth. 

These  media  are  prepared  by  adding  to  tubes  of  ordinary  sterile  broth, 
one-half,  one-third  or  one-quarter  their  volume  of  blood-serum,  ascitic 
fluid,  or  blood  collected  under  aseptic  precautions  (p.  45  and  Chap.  XII.). 

Achalme,  in  the  preparation  of  blood  broth,  advises  the  use  of  a  1  per 
cent,  solution  of  commercial  haemoglobin  instead  of  blood.  The  haemoglobin 
must  first  be  sterilized  by  filtration  through  a  Kitasato's  filter  (p.  25). 

The  preparation  of  these  media  will  be  more  fully  considered  when  dealing 
with  the  Gonococcus  and  Pfeiffer's  bacillus. 

On  account  of  the  difficulty  of  obtaining  sterile  blood,  Bernstein  and  Epstein 
recommend  the  following  procedure  for  the  preparation  of  blood  broth:  collect 
400  c.c.  of  ox  blood  in  a  flask  containing  30  c.c.  of  a  1  per  cent,  solution  of  ammonium 
oxalate  in  distilled  water  and  0*5  c.c.  of  formalin  ;  shake,  and  in  half  an  hour 
dilute  the  mixture  with  3  volumes  of  normal  saline  solution.  After  standing  for  a  day 
or  two  at  the  temperature  of  the  laboratory,  distribute  in  agar  or  broth  in  the  pro- 
portion of  1  part  to  15  of  medium.  The  formalin  is  thus  so  highly  diluted  that  it 
does  not  interfere  with  the  growth  of  micro-organisms.  The  Pneumococcus,  Gono- 
coccus and  Meningococcus  all  grow  very  well  in  this  medium. 

Carbohydrate  broths. 

These  media  are  prepared  by  adding  to  beef  broth,  at  the  "same  time  as 
the  peptone  and  salt,  2-4  per  cent,  of  one  or  other  of  the  following  carbo- 
hydrates :  glucose,  saccharose,  lactose,  galactose,  mannite,  dulcite,  maltose, 
Isevulose  ;  the  preparation  is  completed  as  in  the  case  of  broth. 


ANIMAL   TISSUES   AND   FLUIDS  35 

[The  use  of  carbohydrate  media  has  been  considerably  extended  in  recent 
years  in  connexion  with  the  identification  and  differentiation  of  the  various 
members  of  the  same  group  of  micro-organisms,  e.g.  the  differentiation  of 
members  of  the  typhoid-colon  group,  the  differentiation  of  the  streptococci, 
etc.  (Gordon,  Andrewes  and  Horder  and  others).  For  this  purpose  a  medium 
differing  somewhat  from  that  given  above,  and  having  the  following  com- 
position, is  in  general  use. 

Peptone.  -  1-2  grams. 

Water,  -  -  100  c.c. 

Test  substance,        -  1  gram. 

Kahlbaum's  litmus  solution.  -                                                   -  Q.8. 

[The  test  substance  may  be  either  a  sugar,  e.g.  glucose,  lactose,  Isovulose, 
saccharose,  etc. — an  alcohol,  e.g.  mannite,  dulcite— or  a  glucoside,  e.g.  salicin, 
coniferin,  etc. 

[Care  must  be  taken  to  obtain  guaranteed  pure  chemicals  from  reliable  firms,  and 
an  equal  amount  of  care  must  be  bestowed  upon  the  sterilization  of  the  media,, 
since  it  is  well  known  that  in  the  presence  of  water  and  under  the  influence  of  heat 
many  of  these  highly  complex  compounds  undergo  decomposition,  often  of  the 
nature  of  an  hydrolysis.  Filtration  through  a  porcelain  filter  would  seem  to 
be  the  best  method  of  sterilization.  After  distribution  into  sterile  tubes  the  latter 
must  be  incubated  for  a  few  days  and  those  showing  any  change  rejected.] 

Glycerin  broth. 

Add  5  per  cent,  or  50  grams  per  litre  of  pure  glycerin  to  peptone-beef- 
broth  before  distribution  into  tubes  (Stage  7,  p.  31). 

Glycerin  in  the  same  proportion  may  also  be  added  to  the  carbohydrate 
broths  prepared  as  above. 

Carbonated  broth. 

Add  calcium  carbonate  (2  per  cent.)  x  to  lactose-,  mannite-,  glucose-,  etc., 
broth  before  distributing  into  tubes  (Stage  7,  p  31). 

Calcium  carbonate  is  most  frequently  added  to  lactose- broth.  When  an  organism 
which  ferments  a  given  sugar  is  grown  in  a  carbonated  broth  containing  that  sugar, 
the  acids  formed  by  decomposition  of  the  carbohydrate  act  on  the  chalk  with  the 
formation  of  CO2  and  the  evolution  of  a  considerable  quantity  of  gas.2 

Milk. 

Milk  is  used  as  a  culture  medium  in  several  ways. 

(A)  Fresh  milk,  alkaline  in  reaction,  is  distributed  in  tubes  (15-20  c.c. 
per  tube). 

The  tubes  are  plugged  with  wool,  and  sterilized  at  115°  C.  for  20  minutes. 

This  is  the  most  simple  method  of  preparation  and  suffices  in  the  great 
majority  of  cases  ;  it  is  the  method  ordinarily  employed. 

[In  England  it  is  usual  to  add  sufficient  litmus  solution  to  tint  the  milk 
blue  (p.  57),  and  to  sterilize  by  steaming  at  100°  C.  (Chap.  I.).  In  our 
experience  it  is  a  very  difficult  matter  to  sterilize  milk  by  steam  at  100°  C. 
in  bulk  ;  it  is  much  safer  to  tube  the  milk  and  then  sterilize  it.] 

(B)  Since  a  temperature  of  115°  C.  alters  to  some  extent  the  properties  of 
milk,  it  may  be  desirable  for  some  purposes  to  sterilize  at  a  lower  tempera- 
ture. 

In  that  case,  after  washing  the  cow's  udder  with  an  antiseptic,  the  milker 

[l  In  our  experience  0'5  per  cent.,  or  even  0'25  per  cent,  of  calcium  carbonate  is 
sufficient.] 

[2  It  sometimes  happens,  however,  that  when  an  organism  is  grown  in  a  litmus-sugar- 
carbonate-broth,  acid  is  formed  as  shown  by  the  change  in  colour  of  the  litmus  but  no 
gas  is  evolved.] 


36  LIQUID   MEDIA 

should  sterilize  his  hands  and  then  collect  the  milk  as  it  leaves  the  udder  in 
sterile  flasks.  (For  further  details,  see  Chap.  XII.) 

Each  flask  is  about  three-parts  filled,  sealed  in  the  flame,  [or  plugged  with 
sterile  wool  and  covered  with  an  india-rubber  cap,]  and  heated  in  a  water 
bath  at  60°-65°  C.  for  eight  days  in  the  manner  described  on  p.  12. 

When  sterilization  is  completed,  the  milk  can  be  tubed  into  sterile  tubes, 
as  described  in  connexion  with  the  preparation  of  serum  (p.  45). 

(C)  If  the  technique  of  the  milking  process  can  be  relied  upon,  it  will  be 
sufficient  to  fill  as  many  tubes  as  are  required,  and  to  incubate  them  at  30°  C. 
for  some  days  before  using  the  milk  as  a  culture  medium.  In  spite  of  every 
precaution  some  of  the  tubes  will  be  contaminated,  and  any  tube  in  which 
the  milk  has  clotted  or  which  on  microscopical  examination  shows  the 
presence  of  organisms  must  be  rejected. 

Urine. 

Though  urine  was  widely  employed  in  the  early  days  of  bacteriology,  it 
has  now  almost  ceased  to  be  used  as  a  culture  medium. 

(a)  1.  Boil  some  recently  passed  urine. 

2.  If  the  reaction  be  markedly  alkaline  after  boiling,  add  a  little  tartaric  acid 
solution,  testing  the  reaction  with  litmus  paper. 

3.  Filter,  tube  and  sterilize  at  115°  C. 

The  composition  of  the  urine  is  distinctly  altered  by  this  proceeding,  the  urea 
in  solution  being  decomposed  at  the  temperature  of  boiling  water. 

(6)  It  is  better  to  sterilize  by  filtering  through  a  Chamberland  bougie  (Chap.  I.). 

(c)  To  collect  urine  in  a  sterile  manner,  and  so  avoid  the  necessity  for  steriliza- 
tion with  the  attendant  alteration  in  composition,  proceed  as  in  Chap.  XII.  ("Urine"). 

The  urine  which  has  been  collected  in  a  flask  may  be  tubed  by  any  of  the  methods 
described  for  tubing  serum  (p.  45).  Incubate  the  tubes  at  37°  C.  for  48  hours, 
and  reject  any  which  are  then  cloudy. 

Serum. 

Serum  is  obtained  by  allowing  blood  to  clot  spontaneously  or  from  the 
fluid  of  pleural  effusions.  It  is  used  sometimes  as  a  liquid  but  much  more 
commonly  as  a  solid  medium  after  being  coagulated  by  heat. 

The  technique  for  the  collection  of  serum  will  be  studied  under  the  head 
of  solid  media  (pp.  45  et  seq.}. 

Blood. 

Blood  is  frequently  used  as  a  culture  medium. 

To  use  it  as  a  liquid  medium  coagulation  must  be  prevented,  and  this 
may  best  be  done  by  defibrinating  the  blood.  The  blood  is  collected  asepti- 
cally  (pp.  45  and  48  and  Chap.  XII.)  in  a  sterile  flask  containing  glass  beads 
and  shaken  for  about  10  minutes,  then  aspirated  into  a  [Cobbett's  bulb  or] 
Chamberland  flask  (pp.  45  and  47)  and  tubed. 

Among  the  many  substances  which  it  has  been  suggested  might  be  added  to 
blood  to  prevent  coagulation,  neutral  sodium  citrate  and  extract  of  leeches'  heads 
may  be  mentioned. 

By  the  sodium  citrate  method  the  blood  is  collected  as  it  leaves  the  vein  in  a 
flask  or  tube  containing  a  certain  quantity  of  the  following  sterile  solution  :  water, 
1000  c.c.  ;  sodium  chloride,  8  grams  ;  sodium  citrate,  15  grams. 

Extract  of  leech  heads  is  obtained  by  placing  the  heads  in  75  per  cent,  alcohol 
for  5  or  6  days.  When  hardened,  the  heads  are  dried  and  ground  up  in  a  mortar. 
The  powder  is  dissolved  in  distilled  water  (100  c.c.  per  head),  boiled,  filtered  and 
sterilized  at  105°  C.  for  5  to  10  minutes.  The  extract  is  then  introduced  into 
the  tubes  in  which  the  blood  is  to  be  collected. 

These  last  two  methods  are  not  so  good  as  defibrination. 


VEGETABLE   INFUSIONS  37 

2.  Media  made  from  vegetable  tissues. 

Vegetable  infusions  are  but  seldom  used  in  practical  bacteriology.  The 
most  important  of  them,  however,  may  be  mentioned. 

Malt  extract. 

1.  Grind  up  100  grams  of  germinated  barley  (malt),  and  add  1000  grams 
of  water. 

2.  Heat  the  mixture  to  55°-58°  C.  for  one  hour  :   the  starch  is  converted 
into  maltose  by  the  diastase  and  a  true  beer  wort  obtained  ;  the  tempera- 
ture must  not  exceed  58°  C.,  otherwise  the  diastase  will  be  destroyed. 

3.  Boil.     Filter  through  Chardin  paper. 

4.  Tube.     Sterilize  at  115°  C. 

Yeast  extract. 

Mix  100  grams  of  yeast  with  1000  grams  of  water.  Boil  and  filter  through 
Chardin  paper. 

Tube  the  slightly  acid  filtrate  or  pour  it  into  a  flask,  and  sterilize  at 
115°  C. 

The  filtrate  may  be  neutralized  or  made  slightly  alkaline  by  the  careful  addition 
of  normal  soda  solution  before  filtering.  The  addition  of  5  per  cent,  cane  sugar 
or  glucose  before  filtration  increases  the  nutritive  value  of  the  extract.  If  the 
extract  is  not  clear  when  filtered,  a  little  phosphoric  acid  1  may  be  added,  and  the 
reaction  brought  back  with  lime  water.  Heat  to  116°-117°  C.  for  5  minutes.  Filter. 
Tube.  Sterilize  at  115°  C. 

Spronck's  peptone  yeast  extract. 

1.  To  5  litres  of  water  add  1000  grams  of  commercial  yeast  (not  brewers* 
yeast). 

2.  Boil  the  mixture  for  20  minutes,  stirring  frequently,  pour  into  cylindrical 
vessels  and  leave  for  24  hours. 

3.  Decant  the  cloudy  liquid  and  add  5  grams  of  salt  and  10  grams  of 
Witte's  peptone  for  each  litre. 

4.  Neutralize  exactly,  and  then  make  alkaline  to  the  extent  of  7  c.c.  of  normal 
soda  per  litre.     Boil.     Filter  through  Chardin  paper.2 

5.  Pour  into  flasks.     Sterilize  at  115°-120°  C. 

Hay  infusion.    Straw  infusion. 

Macerate  15-20  grams  of  finely  chopped  hay  or  straw  in  1000  grams  of 
water  for  1  or  2  hours.  Boil  for  a  few  minutes,  filter,  tube  and  sterilize 
at  115°  C. 

The  infusion  which  is  sometimes  a  little  acid  may  be  neutralized  in  the 
ordinary  way. 

Potato  infusion. 

Clean  and  scrape  a  few  potatoes  ;  add  a  litre  of  water  to  each  20-30 
grams  of  pulp.  Leave  to  stand  for  3  or  4  hours.  Decant.  Boil  the 
supernatant  fluid.  Filter.  Tube.  Sterilize.  The  infusion  is  often  acid 
and  can  be  neutralized  before  filtration. 

Infusion  of  carrot  is  prepared  in  a  similar  manner. 

Haricot  decoction. 

1.  Macerate  50-60  grams  of  white  haricot  beans  in  a  litre  of  water  for 
several  hours  in  the  cold. 

1  Specific  gravity  1-349,  and  containing  39 '4  grams  of  anhydrous  acid  per  cent. 

2  If  the  yeast  contain  meal  the  filtered  liquid  remains  slightly  cloudy,  but  this  is  of 
no  consequence. 


38 


LIQUID   MEDIA 


2.  Boil  for  half  an  hour. 

3.  Pour  on  to  a  coarse  sieve,  collect  the  liquid,  and  add  to  it  1  per  cent, 
salt,  2  per  cent,  ordinary  sugar  and  a  pinch  of  sodium  bicarbonate.     Boil. 
Filter  through  paper. 

4.  Tube.     Sterilize  at  115°  C. 

This  medium  is  used  by  Maze  for  the  cultivation  of  the  micro-organism 
found  in  the  nodules  of  leguminous  plants. 

Decoction  of  dried  fruits. 

1.  Macerate  50-100  grams  of  dried  fruits   (prunes   or   raisins)  in  a  litre 
of  water  for  several  hours.     Then  stew  them  in  the  water. 

2.  Pass  through  a  coarse  sieve. 

3.  Boil.     Filter. 

4.  Tube.     Sterilize  at  115°  C. 

The  liquid  is  slightly  acid  and  is  useful  for  cultivating  moulds.  For  other 
purposes  neutralize  with  soda  solution  before  boiling  (Stage  3). 

Wine. 

Wine  was  much  used  by  Pasteur  in  his  early  work,  but  is  now  hardly 
ever  seen  in  the  laboratory.  Before  sterilizing,  neutralize  or  make  slightly 
alkaline  with  soda  solution  in  the  ordinary  way. 


3.  Synthetic  media. 

These  media,  though  seldom  used  in  everyday  work,  have  been  employed 
ior  the  study  of  certain  problems  in  the  biology  of  micro-organisms. 

The  formulse  of  the  best  known  are  given  below.  Some  others  will  be 
described  in  connexion  with  the  organisms  in  the  study  of  which  they  have 
been  employed. 

Pasteur's  medium. 

Water,  -  100  grams. 

Candied  sugar,          -  -         10       ,, 

Ammonium  tartrate.  -  O'lOgram. 

Ash  of  yeast,  -  0'075    „ 

Boil.     Filter.     Tube.     Sterilize.     The  reaction  is  alkaline. 


Raulin's  medium. 


Water,  - 
Candied  sugar, 
Tartaric  acid, 
Ammonium  nitrate, 
Ammonium  phosphate, 
Potassium  carbonate, 
Magnesium  carbonate, 
Ammonium  sulphate, 
Zinc  sulphate, 
Sulphate  of  iron, 
Potassium  silicate, 


-     1500  grams. 
70 
4 
4 

0P6  gram. 
0-6 
0-4 
0-25 
0-07 
0-07 
0-07 


Prepare  as  in  the  case  of  Pasteur's  medium. 

The  reaction  is  acid.     This  medium  was  used  by  Raulin  in  his  well-known 
work  on  Aspergillus  niger. 

Cohn's  medium. 


Distilled  water, 
Ammonium  tartrate, 
Potassium  phosphate, 
Magnesium  sulphate, 
Tricalcium  phosphate, 


200  grams. 
2 
1  gram. 

O'lO  " 


Prepare  as  in  the  case  of  Pasteur's  medium.     The  reaction  is  alkaline. 


SYNTHETIC  MEDIA 


39 


Water,  - 

Ammonium  tartrate, 
Potassium  phosphate, 
Magnesium  sulphate, 
Calcium  chloride,     - 

Prepare  as  above. 


Nsegeli's  medium. 


Distilled  water, 
Glycerin, 
Sodium  chloride, 
Calcium  chloride,     - 
Magnesium  sulphate, 
Di-potassium  phosphate, 
Ammonium  lactate, 
Potassium  aspartate, 


1000  grams. 
10 

1     gram. 
0-2     „ 
0-12   „ 


Uschinsky's  medium. 

1000  c.c. 
30  grams. 
5 

O'l  gram. 
0-2     „ 

2  grams. 
6 

3  rj 

The  method  of  preparation  is  the  same  as  in  the  other  cases.     This  medium 
was  used  by  Uschinsky  in  his  work  on  diphtheria  toxin. 


SECTION   II.— SOLID   MEDIA. 

The  introduction  of  solid  media  into  practical  bacteriology  is  due  to 
Schroeter  and  especially  to  Koch.  The  commonest  are  transparent  media, 
prepared  by  adding  to  broth  substances  capable  of  making  it  solid  at  ordinary 
temperatures  ;  but  albumins  coagulated  by  heat  (serum,  egg,  etc.),  meat 
and  certain  vegetable  media  are  also  used. 


1.  Gelatin  media. 

Gelatin  media  are  in  very  general  use,  and  several  different  sorts  are 
prepared. 

General  rules. — 1.  Use  extra  quality  French  gelatin,  which  is  sold  in  thin 
rectangular  sheets  weighing  about  2*5  grams  each.  (Ordinary  commercial 
gelatin  loses  its  property  of  solidifying  if  heated  above  102°-105°  C.  and 
sterilization  must  therefore  be  effected  at  100°  C.  ;  this  introduces  an 
unnecessary  complication  into  the  preparation  of  the  medium.) 

2.  Gelatin  is  very  acid,  and  the  medium  must  be  neutralized  after  adding 
it  to  the  other  constituents,  but  the  addition  of  alkali  must  be  stopped  at  the 
neutral  point  or  when  the  reaction  is  very  slightly  alkaline,  because  gelatin 
will  not  solidify  after  being  heated  in  alkaline  solution. 

3.  Ordinary"  gelatin  media  liquefy  at  25°  C.,  and  can  therefore  only  be 
used  when  the  temperature  of  incubation  is  not  to  exceed  20°-23°  C. 

Ordinary  gelatin. 

This  medium  is  generally  known  simply  as  gelatin. 
Method  recommended. — Proceed  as  in  the  preparation  of  broth. 
1,  2  and  3.  Macerate  500  grams  of  lean  beef  in  a  litre  of  water,  heat,  express 
the  fluid,  filter  while  hot  and  make  up  the  volume  to  a  litre. 

4.  To  this  broth  add 


10  grams. 
5 

a  pinch  (not  essential). 
80-150  grams. 


Peptone  (Chapoteaut), 

Salt, 

Sodium  phosphate, 

Extra  quality  gelatin, 
The  amount  of  gelatin  required  varies  according  to  the  time  of  year  :    in 
winter  8  per  cent.  (80  grams  per  litre)  is  sufficient,  but  in  summer  as  much  as 
10  to  15  per  cent,  is  necessary — say  120  grams  per  litre. 


40 


SOLID   MEDIA 


Warm  the  mixture  at  a  gentle  heat  in  an  enamelled  saucepan,  stirring 
constantly  to  prevent  the  gelatin  sticking  to  the  bottom.  When  the  gelatin 
is  dissolved,  boil  for  two  or  three  minutes. 

5.  The  medium  is  now  very  acid  ;   add  soda  solution  carefully,  testing  the 
reaction  with  litmus  paper  after  each  addition.     The  end  reaction  should  be 
neutral  or  very  slightly  alkaline. 

6.  Autoclave  for  5  minutes  at  115°  C.  in  a  flask  or  enamelled  vessel,  to 
precipitate  earthy  phosphates. 

7.  On  taking  out  of  the  autoclave  pour  the  hot  fluid  on  to  a  moistened 
Chardin  paper  fixed  in  a  hot  water  funnel :    the  filtration  must  be  done  in 

the  warm,  otherwise  the  gelatin  will  solidify  before  it 
has  filtered. 

A  more  simple  method  consists  in  filtering  through  a 
glass  funnel  fitted  into  a  flat- bottomed  flask,  the  flask  and 
funnel  being  placed  in  the  autoclave  [or  steamer],  which  is 
heated  to  100°  C.  Filtration  is  quite  easy  under  these  con- 
ditions. 

Hot  water  funnel. — This  piece  of  apparatus  consists  of  a 
copper  funnel  mounted  on  legs  (fig.  32),  and  lined  by  a  second 
— glass — funnel,  the  delivery  tube  of  which,  passing  through 
the  neck  of  the  metal  funnel,  is  made  to  fit  it  closely  with  an 
india-rubber  bung.  Pour  water  into  the  space  between  the 
two  funnels  through  a  small  lateral  opening  provided  for  the 
purpose,  and  heat  the  apparatus  by  means  of  a  Bunsen 
burner  placed  beneath  a  side  tube  projecting  from  the  lower 
part  of  the  metal  funnel.  The  temperature  should  not  be 
raised  to  boiling  point,  otherwise  the  water  will  be  driven 
out  through  the  inlet  tube.  Heat  the  funnel  before  pouring 
the  gelatin  on  it.  Several  patterns  of  this  apparatus  are 
made ;  one  useful  form  is  that  in  which  the  metal  funnel  has 
two  metal  walls,  and  the  glass  funnel  fits  inside  the  inner  wall. 

8.  Collect  the  filtered  liquid  in  a  flask  and  tube  it  at  once — before  it  has 
had  time  to  solidify — in  quantities  of  10-15  c.c.  in  each  tube. 

A  small  glass  funnel  should  always  be  used  for  tubing  to  avoid  soiling  the  mouth 
of  the  tubes,  as  has  been  already  explained  (p.  32). 
The  medium  should  be  perfectly  clear. 

9.  Plug  the  tubes  :    sterilize  at  110°  C.  for  20  minutes,  taking  care  that 
the   temperature   does    not    reach    115°  C.     [Sterilization    may  be  effected 
equally  by  heating  in  the  steamer  to  100°  C.  for  20  minutes  on  each  of  three 
successive  days.] 

Notes. — (a)  Gelatin  prepared  in  this  way  is  quite  clear  and  absolutely  trans- 
parent. If  the  liquid  be  slightly  cloudy  after  filtering,  add  the  white  of  an  egg 
beaten  up  in  50  to  100  c.c.  of  water,  mix  thoroughly  and  autoclave  for  5  minutes 
at  115°  C.  ;  then  after  filtering  through  Chardin  paper  the  mixture  is  perfectly 
clear.  It  is  advised  that  this  method  be  employed  as  seldom  as  possible  as  it  is 
not  without  influence  on  the  melting  point  of  the  gelatin. 

(6)  The  low  temperature  at  which  gelatin  melts  (23°- 25°  C.)  being  a  disadvantage 
in  the  use  of  the  medium,  bacteriologists  have  attempted  to  raise  the  melting  point 
by  modifying  the  method  of  preparation.  Many  of  these  modifications  seem  to 
be  of  no  practical  value :  the  author  has  never  found  any  advantage  in  using  car- 
bonate of  soda  for  neutralization  as  recommended  by  Bertarelli,  and  similarly 
Roux's  method  of  sterilization  at  100°  C.  on  several  successive  occasions  appears 
uselessly  to  complicate  the  preparation  of  the  medium  without  offering  any  cor- 
responding advantage. 

By  following  the  instructions  given  above,  and  provided  that  (1)  a  good  quality 
of  gelatin  be  used,  (2)  neutralization  be  stopped  at  the  neutral  point  or  when  the 
liquid  is  very  slightly  alkaline,  and  (3)  a  temperature  of  115°  C.  be  never  exceeded, 
a  10  per  cent,  gelatin  is  easily  obtained  which  does  not  melt  below  25°  C.  ;  and 


FIG.  32. — Hot  water 
funnel. 


GELATIN  MEDIA  41 

by  raising  the  gelatin  content  to  15   per  cent,  the  medium  will  remain  solid  at 
summer  temperature. 

An  important  point  is  to  cool  the  tubes  rapidly  on  taking  them  out  of  the  auto- 
clave, and  to  store  them  in  a  cold  place  as  recommended  by  Abba. 

Fischer's  gelatin. 

For  the  cultivation  of  phosphorescent  bacteria  Fischer  recommends  a 
gelatin  very  rich  in  sodium  chloride.  To  a  litre  of  meat  extract  prepared 
as  above  add 

Peptone  (Chapoteaut),      -  10  grams. 

Salt,       -  30 

Gelatin  (extra  quality),    -  80-120 

Dissolve,  and  proceed  as  in  the  preparation  of  ordinary  gelatin. 

Liebig's  gelatin. 

Dissolve  5  grams  of  Liebig's  extract  [Lemco]  in  1000  grams  of  water  (add 
if  necessary  10  grams  of  peptone,  and  5  grams  of  salt),  then  add  100  grams 
of  gelatin,  dissolve,  and  boil  for  2  or  3  minutes.  Neutralize  and  complete 
the  preparation  as  for  ordinary  gelatin. 

Buchner's  gelatin. 

1.  Dissolve  with  heat  in  a  litre  of  water 

Gelatin  (extra  quality),    -  -       100  grams. 

Cane  sugar,     -  -         20         „ 
Liebig's  extract  [Lemco],  5         ., 

Dry  peptone,  -  5         ,, 

2.  To  the  solution  add 

Tricalcium  phosphate,     -  5  grams. 

3.  Boil  for  a  few  minutes,  heat  to  115°  C.,  filter  and  proceed  in  the  ordinary 
way. 

Raisin  gelatin. 

1.  Make  a  decoction  as  described  on  p.   38,  consisting  of  250  grams  of  dried 
raisins  in  a  litre  of  water. 

2.  Filter,  then  add  100  grams  of  gelatin  and  a  pinch  of  sodium  phosphate.     Boil 
for  2  or  3  minutes.     Neutralize  and  finish  as  usual. 

Eisner's  potato  medium 

1.  Take  500  grams  of  potatoes,  peel  and  grate  them. 

2.  Macerate  the  pulp  in  a  litre  of  water  for  3  or  4  hours. 

3.  Strain.     Stand  overnight.     Decant  the  fluid. 

4.  Make  up  the  volume  of  fluid  to  a  litre,  and  dissolve  in  it  with  gentle 
heat   15   to   20   per   cent.    (150  to  200  grams)  of  gelatin.     Boil  for  a  few 
minutes. 

5.  The   fluid  is   now  very   acid.     Add   normal   soda   solution    until    the 
reaction  is  feebly  but  still  distinctly  acid. 

6.  Heat  to  115°  C.  for  5  minutes.     Filter  and  complete  the  preparation 
in  the  ordinary  way. 

Choquet's  gelatin. 

Choquet  recommends  the  two  following  media  for  the  cultivation  of  the 
micro-organisms  concerned  in  dental  caries. 

1.  Meat  extract,  500  c.c. 

Gelatin  (extra  quality  white),   -  -         35  grams. 

Peptone  (Chapoteaut),      -  5         „ 

Calcium  glycero-phosphate,       -  5         ,, 


42 


SOLID   MEDIA 


2.  Meat  extract,  500  c.c. 

Peptone,          -  5  grams. 

Gelatin,  35 

Calcium  phosphate,  50         ,, 

Magnesium  phosphate,  5         „ 

Calcium  carbonate,  10         ., 

Dissolve  the  gelatin  and  peptone  in  the  meat  extract :  heat  in  the  auto- 
clave and  filter  in  the  ordinary  way.  After  filtration  and  before  distributing 
in  tubes  add  the  calcium  and  magnesium  salts. 

2.  Agar  media. 

Agar-agar  is  derived  from  a  sea-weed  growing  in  the  Indian  Ocean,  and 
in  commerce  occurs  as  dried  fibrous  strands. 

When  agar  is  boiled  with  water  it  forms  a  firm  jelly  which  does  not  melt 
below  90°  C.  Agar  is  therefore  substituted  for  gelatin  whenever  a  solid 
medium  is  required  for  incubation  above  25°  C. 

The  preparation  of  agar  media  is  tedious  because  agar  readily  forms  with 
water  a  thick  jelly  which  is  difficult  to  filter.  This  difficulty  is  overcome  by 
altering  the  properties  of  the  agar  by  prolonged  boiling  or  by  chemical 
action,  e.g.  the  addition  of  acid. 

Another  difficulty  arises  from  the  fact  that  agar  is  always  cloudy  if  not 
cleared  with  albumen,  and  even  then  it  is  sometimes  opalescent. 

Ordinary  Agar. 

An  agar  medium  prepared  according  to  the  method  now  to  be  described 
is  generally  spoken  of  as  agar,  and  the  word  will  be  so  used  in  this  book. 

Preparation  of  Agar. 

[A.  Method  recommended. — 1.  Weigh  out  30  grams  of  agar  fibre,  turn  it 
into  a  2-litre  flask  and  fill  the  flask  nearly  full  of  tap  water,  then  add  10  c.c. 
of  a  2  per  cent,  solution  of  acetic  acid  and  stir  well  with  a  glass  rod. 

[2.  Leave  the  agar  to  soak  for  10  minutes,  then  put  a  large  funnel  into  the 
flask,  stand  the  funnel  under  the  cold  water  tap  and  wash  the  agar  in  running 
water  until  the  washings  are  neutral  to  litmus  paper  (10  minutes). 

[3.  While  preparing  the  agar,  stand  a  flask  containing  a  litre  of  broth  in 
the  steamer  and  heat  to  100°  C. 

[4.  Add  the  washed  agar  to  the  hot  broth. 

[5.  Heat  the  mixture  in  the  steamer  at  100°  C.  until  the  agar  is  dissolved 
(20  minutes). 

[6.  The  medium  is  now  a  little  acid.  Neutralize  with  a  10  per  cent,  solution 
of  caustic  soda  (about  1  c.c.)  and  allow  the  contents  of  the  flask  to  cool  to 
-50°-60°  C. 

[7.  Beat  up  the  white  of  two  eggs  in  a  beaker,  and  add  to  the  cooled  agar. 
Mix  thoroughly. 

[8.  Heat  the  mixture  in  the  steamer  at  100°  C.  until  the  egg-albumin  is 
coagulated,  and  until  on  holding  up  the  flask  to  the  light  the  agar  is  clear  (f-1 
hour).  At  the  same  time  put  into  the  steamer  a  Chardin  filter  paper  arranged 
in  a  funnel,  the  latter  standing  in  a  sterile  flask. 

[9.  When  the  medium  is  clear — there  will  nearly  always  be  lumps  of  coagu- 
lated albumin  floating  about  in  the  agar — pour  it, on  to  the  hot  filter  in  the 
.steamer.  The  filter  must  not  be  taken  out  of  the  steamer,  and  the  medium 
should  be  poured  down  the  sides  of  the  filter  paper. 

[10.  Place  the  lid  on  the  steamer,  and  maintain  the  heat  until  the  medium 
lias  all  filtered  through  (15  minutes). 


AGAR   MEDIA  43 

[11.  Tube,  and  sterilize  for  30  minutes  at  100°  C.  on  two  successive 
days.  Slope  (see  B.  9  infra}. 

[This  method  always  gives  a  perfectly  clear,  transparent  and  very  slightly 
opalescent  medium.  The  yield  is  very  approximately  100  per  cent. — that  is 
to  say  that  from  a  litre  of  broth  rather  more  than  a  litre  of  agar  is  obtained. 
No  trouble  is  ever  experienced  in  getting  the  agar  to  adhere  to  the  walls  of 
the  tubes. 

[If  the  autoclave  be  used  the  medium  is  generally  of  a  brownish  colour 
from  over-heating,  and  it  is  sometimes  difficult  to  get  the  medium  to  stand 
up  in  the  tubes.] 

B.  Another  method,  which  is  also  recommended,  is  as  follows  : 

1.  Prepare  a  peptone-beef-broth,  according  to  the  instructions  given  on 
p.  30,  up  to  and  including  Stage  5. 

2.  To  this  broth  add  20  grams  (2  per  cent.)  of  agar  cut  up  into  small 
pieces.     The  agar  should  be  swollen  by  soaking  in  cold  water  for  an  hour 
or   two,    and    then   wrung   out   in   a    cloth    before    being    added    to    the 
broth. 

3.  Heat  the  mixture  to  100°  C.  in  an  enamelled  saucepan,  and  keep  it  at 
this  temperature  until  the  agar  is  dissolved  (about  half  an  hour),  stirring 
all  the  time. 

4.  Test  the  reaction,   which   should   be   neutral  or  faintly  alkaline.     If 
heated  in  presence  of  acid,  agar  becomes  converted  into  sugar. 

5.  Cool  to  55°  or  60°  C.  and  add  the  white  of  an  egg  beaten  up  in  100  grams 
of  water.     Mix  thoroughly. 

6.  Autoclave  for  an  hour  at   120°  C.     The  albumin  is  coagulated   and 
carries  down  the  impurities  with  it. 

7.  Pour  the  liquid  while  still  hot  on  to  a  moistened  Chardin  paper  arranged 
in  a  hot  water  funnel.     Cover  the  funnel  with  a  glass  plate. 

8.  Collect  the  liquid  as  it  niters  in  a  previously  sterilized  flask,  and  tube 
at  once.     This  must  be  done  as  quickly  as  possible  as  agar  sets  about  40°  C., 
and  a  funnel  as  usual  should  be  used  in  tubing  it  to  prevent  the  medium 
soiling  the  mouths  of  the  tubes.     Each  tube  should  contain  8  to  10  c.c. 

9.  Sterilize  at  115°  C.  for  20  minutes.     After  sterilization  and  while  the 
medium  is  still  hot,  slope  the  tubes  on  some  such  piece  of  apparatus  as  that 
pictured  on  p.  52,  so  that  the  agar  solidifies  with  a  sloped  surface.     Leave  the 
tubes  in  this  position  for  36  hours. 

Some  bacteriologists  recommend  the  addition  of  a  small  quantity  of  an  aqueous 
solution  of  gum  arabic  to  the  agar,  to  prevent  the  thin  upper  part  becoming  detached 
from  the  wall  of  the  tube  when  it  is  placed  vertically.  But  gum  arabic  makes  the 
medium  distinctly  cloudy,  and  does  not  appear  to  effect  the  purpose  for  which  it 
is  added. 

If  the  method  of  preparation  described  be  followed  step  by  step,  the  agar  will 
be  found  to  adhere  sufficiently  well.  Gelatin  to  the  amount  of  20  grams  per  litre 
may  be  added  as  recommended  by  Nicolle. 

Modification.— Filtration  may  be  accomplished  in  the  following  manner, 
even  more  easily  than  by  the  above  method. 

Before  adding  the  agar  to  the  broth  (Stage  2)  leave  it  to  soak  in  6  per 
cent,  hydrochloric  acid  (water,  500  :  HC1,  30)  for  24  hours,  and  wash  m  a 
large  quantity  of  water.  Then  soak  in  a  5  per  cent,  solution  of  ammonia 
(water,  500  :  ammonia,  25)  for  some  hours,  wash  in  a  large  quantity  of 
water,  and  squeeze  the  agar  dry  in  a  cloth.  The  agar  is  now  ready  to  add 
to  the  broth,  and  the  further  steps  are  as  described.  The  resulting  jelly 
does  not  adhere  well  to  the  walls  of  the  tubes,  and  the  process  cannot  be 
recommended. 


44  SOLID   MEDIA 

Karlin ski's  filter. — To  facilitate  filtration,  Karlinski  has  devised  an  apparatus  in  which 
the  agar  is  filtered  under  pressure.  A  water-jacketed  copper  vessel  heated  below  by  a 
ring  Bunsen  is  fitted  with  a  copper  cylinder,  the  bottom  of  which  shaped  like  a  funnel 
and  terminating  in  a  delivery  tube  fitted  with  a  tap  is  covered  with  a  layer  of  absorbent 
wool.  The  agar  is  poured  on  to  the  wool,  and  the  upper  opening  is  hermetically  sealed 
by  means  of  a  cover  through  which  the  tube  of  an  india-rubber  syringe  passes.  When 
the  ball  of  the  syringe  is  squeezed,  the  air  in  the  apparatus  above  the  agar  is  compressed 
and  forces  the  agar  through  the  wool.  This  ingenious  piece  of  apparatus  does  not  appear 
essential  because  if  the  agar  be  prepared  in  either  of  the  ways  described  no  difficulty  will 
be  experienced  in  filtering  through  Chardin  paper. 

Fischer's  method. — Fischer  proposes  to  overcome  the  difficulty  of  filtration  as  follows. 
Plug  the  narrow  end  of  a  funnel  with  an  ordinary  cork,  and  pour  the  agar  into  the  funnel 
at  once  on  taking  it  out  of  the  autoclave.  Allow  to  cool.  The  solid  particles  which 
cause  the  cloudiness  settle  to  the  bottom  of  the  funnel.  When  the  agar  is  set  the  jelly 
is  turned  out  whole  and  the  opaque  conical  part  cut  off  with  a  knife.  Cut  up  the  remainder 
into  small  pieces  and  put  into  tubes.  Plug  and  sterilize  the  tubes.  The  resulting  agar 
is  always  opaque. 

Malm's  Agar. 

Add  2  per  cent,  of  agar  to  Liebig's  or  Cibils'  broth  (p.  34).  Proceed  as  for 
ordinary  agar. 

Peptone-agar  (Salomonsen). 

1.  Make  a  broth  with 

Water,  -  -     1000  grams. 

Liebig's  extract,  -  5         „ 

Peptone,          -  30 

Cane  sugar,     -  5         ,, 

If  necessary  add  a  little  alkali. 

2.  Dissolve  15  grams  of  agar  in  the  broth,  and  proceed  as  above. 

Glycerin-agar. 

Add  2  per  cent,  of  agar  to  glycerin  broth  (p.  35),  and  proceed  in  the 
ordinary  way. 

Glucose-glycerin-agar. 

Prepare  a  glucose  broth  (p.  34),  and  after  neutralization  add  5  per  cent, 
neutral  glycerin  and  2  per  cent.  agar.  Complete  the  preparation  in  the 
usual  manner. 

Gelatin-agar. 

By  mixing  agar  and  gelatin  a  medium  is  obtained  the  melting  point  of 
which  lies  between  that  of  agar  and  that  of  gelatin.  In  warm  climates,  in 

the  summer,  agar-gelatin  may  be  used  in  place  of  gelatin.     But  it  must  be 

borne  in  mind  that  the  cultural  characteristics  of  micro-organisms  are  far 

from    being    identical    on    the    two    media.      Gelatin-agar   is    prepared  as 
follows. 

1.  To  1000  grams  of  peptone-broth,  add 

Gelatin,  -         80  grams. 
Agar,      -  5 

or 

Gelatin,  -         50  grams. 
Agar,     -  8         ., 

Dissolve  the  gelatin  in  the  broth,  neutralize,  and  then  add  the  agar. 

2.  Complete  the  preparation  as  in  the  case  of  ordinary  agar,  but  at  Stage  5 
do  not  let  the  temperature  exceed  115°  C. 

Iceland  moss. 

Some  workers  use  Iceland  moss  (Lichen  crispus)  in  place  of  agar,  but  this  sub- 
stitution cannot  be  recommended. 


ALBUMINOUS   MEDIA 


45 


3.  Media  made  from  albuminous  fluids  and  tissues. 

Serum. 

Serum  is  the  liquid  which  separates  when  blood  has  clotted.  In  bacteriology 
bovine,  sheep  and  horse  serum  are  principally  used.  Serum  is  most  frequently 
used  after  coagulation  by  heat,  very  rarely  in  the  liquid  condition. 

An  important  point  about  serum  media  is  that  they  should  be  almost 
transparent,  hence  they  cannot  be  heated  to  a  high  temperature  because 
they  coagulate  en  masse  and  become  opaque.  Liquid  serum  ought  net  to 
be  heated  above  56°  or  58°  C.,  and  to  preserve  its  transparency  solidified 
serum  should  be  coagulated  at  about  70°  C. 

Serum  cannot  therefore  be  sterilized  in  the  ordinary  way. 

Either  (a)  it  must  be  sterilized  by  pasteurization  combined  with  tyn- 
dallization  (Koch's  method)  or  by  filtration  through  a  bougie  :  or  (b)  since 
the  blood  in  the  body  is  sterile,  a  sterile  medium  can  be  obtained  if  care  be 
taken  to  avoid  introducing  contaminations  while  collecting  the  blood  and 
drawing  off  the  serum  (Roux  and  Nocard's  method). 

Collection  of  serum. 
1.    In  the  slaughter-house. 

[A.  Method  recommended. — When  the  blood  is  collected  in  the  slaughter- 
house, the  following  is  a  simple  method  of  proceeding. 

[1.  When  the  carotid  is  severed  discard  the  first  spurt  of  blood,  then  take 
the  plug  out  of  a  sterile  2-litre  flask  and  hold  it  so  that  the  blood  pours 
into  the  open  mouth  :  collect  enough  blood  to  three-parts  fill  the  flask  : 
replace  the  plug.  Collect  as  many  flasks  of  blood  as  are  required. 

[2.  On  reaching  the  laboratory  place  the  flask  on  a  cork  ring,  inclining  it 
as  much  as  possible.  Take  out  the  wool  plug,  burn  the  mouth  of  the  flask 
with  a  Bunsen  burner  both  inside  and  outside  and  plug  the  flask  at  once 
with  clean  wool  ready  sterilized  and  wrapped  in  paper.  Then  stand  the 
flask  vertically,  shaking  it  as  little  as  possible. 


FIG.  33.— Cobbett's  bulb  as  used  for  decanting  serum. 

[3.  When  the  clot  has  formed  and  the  serum  separated,  tilt  the  flask  again 
—the  clot  should  adhere  to  the  bottom— and  introduce  a  piece  of  glass 
tubing  connected  to  a  Cobbett's  bulb  (fig.  33). 


46 


SOLID   MEDIA 


FIG.  34.-  Chamber-land  distri- 
buting flask. 


[The  glass  tubing  is  a  piece  of  ordinary-sized  glass  tubing  bent  near  the 
end  into  an  obtuse  angle  and  sealed.  About  half  a  centimetre  from  the 
sealed  end  a  small  hole  is  blown  into  it  through  which  the  serum  enters. 
This  is  connected  with  the  Cobbett's  bulb  by  a  fairly  long  piece  of  india- 
rubber  tubing. 

[4.  By  aspirating  through  the  plugged  tubulure  the  serum  can  be  drawn 
into  the  bulb.  When  the  bulb  is  nearly  full,  clip 
the  tubing  between  the  flask  and  the  bulb. 

[5.  Remove  the  test-tube  enclosing  the  delivery- 
tube,  and  by  releasing  the  clip  above  it  draw  off 
the  serum  into  test-tubes. 

[6.  Coagulate  the  serum  (p.  51). 
[7.  Incubate  the  tubes  for  48  hours  and  reject 
those  which  show  any  growth.] 

[For  many  purposes,  e.g.  the  cultivation  of  the 
diphtheria  bacillus,  the  serum  may  be  further 
sterilized  after  coagulation  by  heating  it  to  100°  C. 
in  the  steamer  (Chap.  I.). 

[If  the  serum  be  wanted  in  the  liquid  form  for 
future  use,  it  is  best  to  distribute  it  in  small  quan- 
tities into  tubes,  to  heat  it  to  55°-60°  C.  in  the  water 
bath  for  some  time,  and  then  to  add  a  drop  or  two  of  chloroform  to  each 
tube  with  a  sterile  pipette.  The  chloroform  is  readily  driven  off  subsequently 
by  heating  the  tubes  to  40-45°  C.] 

B.  Koch's  method.    Apparatus  required. — Prepare  beforehand  : 

1.  Three  or  four  large  covered  glass  dishes  each  consisting  of  two  halves 
fitting  one  into  the  other,  and  capable  of  holding  2  litres. 

Wrap  the  dishes  in  paper  and  sterilize  them  in  the  hot 
air  sterilizer  at  180°  C.  The  temperature  must  be  raised 
slowly  to  avoid  cracking  the  glass. 

2.  Chamberland  distributing  flasks  (fig.  34). 

Wash  and  dry  the  flasks,  carefully  seal  the  pointed 
tubulure  A  in  the  flame,  plug  the  other  tubulure  B  with 
wool  between  the  constrictions,  and  sterilize  at  180°  C. 

3.  Half-litre  flasks  with  long  necks  (fig.  35).     Plug  and 
sterilize  at  180°  C. 

4.  Sterile  plugged  test-tubes. 

Technique. — 1.  Collect  the  blood  at  the  slaughter-house, 
preferably  in  cool  weather,  in  the  sterilized  glass  dishes. 
To  collect  the  blood  remove  the  dishes  from  their  paper 
wrappings,  and  when  the  beast  is  being  bled,  after  letting 
the  blood  which  first  issues  flow  away,  raise  the  cover  of 
one  of  them  and  collect  enough  blood  to  fill  it  three-parts 
full :  then  replace  the  cover.  Several  dishes  should  be  filled 
in  the  same  way. 

2.  Put  the  dishes  containing  the  blood  in  a  cool  place, 
but  not  in  the  ice  chest,  because  haemolysis  may  occur  and 
so  impart  a  red  colour  to  the  serum. 

3.  After  about  36  hours  the  clot  will  have  formed  and 

shrunk  leaving  the  serum  as  a  clear  fluid  on  top.  Break  off  the  fine  point 
of  a  Chamberland  flask  (fig.  34),  pass  it  through  the  flame  of  a  spirit  lamp 
and  avoiding  all  sources  of  contamination  as  far  as  possible  aspirate  the 
serum  into  the  flask.  Seal  the  point  in  the  flame. 


FIG.  35.— Flask  with 
neck  drawn  out  and 
sealed. 


SERUM  47 

Modification  recommended. — A  better  yield  of  serum  is  obtained  if  instead  of 
collecting  the  blood  in  glass  dishes  Latapie's  apparatus  be  used  (p.  50).  For  the 
present  purpose  the  tube  a  (fig.  40)  is  replaced  by  a  sterile  funnel  by  means  of  which 
the  blood  is  collected  as  it  spurts  from  the  severed  vessel.  When  the  bottle  is  half- 
filled,  the  funnel  is  taken  out  and  an  india-rubber  plug  put  in  its  place.  The  further 
steps  are  described  at  p.  50. 

The  serum  is  sterilized  in  the  following  manner  : 

4.  Take  the  Chamberland  flasks  containing  the  serum  to  the  laboratory. 
In  spite  of  the  precautions  taken  the  serum  will  be  certain  to  be  more  or 
less  contaminated,  and  must  therefore  be  sterilized.     Distribute  it  first  into 
flasks  with  long  necks  thus  :  flame  the  mouth  of  the  flask  in  a  Bunsen  burner 
and  remove  the  cotton-wool  plug  :  break  off  the  capillary  point  of  the  tubulure 
on  the  Chamberland  flask,  pass  the  broken  end  through  the  flame,  and  intro- 
duce it  some  distance  into  the  neck  of  the  other  flask  ;    then  by  blowing 
through  the  tube  B  the  serum  can  be  transferred  to  the  other  flask.     Mean- 
while the  wool  plug  of  this  flask  is  held  between  the  thumb  and  index  finger 
of  the  left  hand. 

5.  The  flask  being  about  three-parts  filled,  its  wool  plug  is  removed  and 
the  neck  heated  in  the  blow-pipe  and  sealed  a  few  centimetres  from  the 
bulb.     As  many  flasks  are  used  as  are  necessary  to  contain  the  serum  collected. 

6.  The  flasks  after  filling  and  sealing  are  heated  in  a  water  bath,  as  already 
described  (p.  12),  to  56°  or  58°  C.  for  one  hour  on  eight  consecutive  days. 

7.  When  sterilized  the  serum  has  to  be  distributed.     A  mark  is  made  on 
the  neck  of  the  flask  with  a  glass  cutter  near  the  sealed  end  and  to  this  scratch 
the  end  of  a  very  hot  glass  rod  is  applied : 

this  cracks  the  glass  and  the  crack  is  ex- 
tended by  touching  the  end  of  it  with  the 
heated  glass  rod.  The  two  ends  of  the 
fracture  soon  join,  and  with  a  gentle  tap 
the  end  of  the  neck  which  was  sealed  in  the 
flame  can  be  easily  separated  and  the  flask 
opened. 

Place  the  flask  B  on  a  cork  ring  E  so  that 
the  neck  is  as  nearly  horizontal  as  possible. 

Flame  the  pointed  tubulure  of  a  sterile 
Chamberland  flask  A  in  a  Bunsen,  break  off 
the  end  with  sterile  forceps  and  insert  the 
tube  into  the  flask  so  as  to  almost  touch 
the  clot  D,  and  aspirate  the  serum  C. 
Discard  the  top  layer  of  serum  because 

having  been  in  contact  with  the  air,  it  may          FIG  36.— Distribution  of  serum, 
possibly  be  contaminated  by  dust  (fig.  36). 

8.  By  means  of  the  Chamberland  flask,  distribute  the  serum  in  sterile  tubes, 
passing  the  mouth  of  each  tube  rapidly  through  the  flame  before  taking  out 
the  wool  plug,  and  flaming  also  the  pointed  tubulure  of  the  Chamberland. 
Pass  the  tapering  end  well  into  the  test-tube,  and  pour  about  10  c.c.  into  each. 
Replace  the  plug  in  the  tube. 

The  serum  is  now  ready  either  for  coagulation  or  for  use  in  the  liquid  state 
(in  the  latter  case  it  should  be  first  incubated  for  48  hours  at  30°  C.). 

If  the  serum  is  to  be  set  this  should  be  done  as  soon  as  possible,  for  should 
any  organisms  have  gained  access  to  the  tubes  during  filling,  the  heat  of 
coagulation  will  very  probably  destroy  them. 

Note. The  serum  may  be  sterilized  by  filtration  instead  of  by  heat ;  but  filtra- 
tion of 'serum  is  a  long  and  tedious  operation,  and  usually  a  troublesome  one,  as  the 


48 


SOLID   MEDIA 


serum  froths  considerably  as  it  comes  through  the  bougie.  If  filtration  be  decided 
upon,  a  Berkefeld  bougie  through  which  the  serum  passes  much  more  readily  than 
through  the  finer  Chamberland  F  type  should  be  used.  The  technique  is  described 
at  pp.  18  et  seq. 

To  facilitate  the  filtration  of  serum  Miquel  has  devised  an  apparatus  which  works 
at  40°  C.  The  serum  is  poured  into  a  cylindrical  vessel  containing  a  filtering  bougie. 
The  cylinder  with  its  contained  bougie  is  placed  in  a  double-walled  vessel  heated  below 
by  a  gas  burner  on  which  a  regulator  is  placed.  The  bougie  is  connected  by  means 
of  india-rubber  tubing  to  a  conical  flask  with  a  tubulure  attached  to  a  water  pump. 
Before  use  the  flask  is  sterilized  in  the  hot  air  sterilizer  and  the  bougie  and  rubber  con- 
nexions in  the  autoclave.  The  side  tube  of  the  flask  should  be  plugged  with  wool. 

2.    From  a  living  animal. 

[A.  Method  recommended. — When  a  living  animal — horse  or  bovine — is 
available,  the  technique  will  be  as  follows. 

[1.  Take  three  or  four  large  (2-3-litre)  sterile  Jena  flasks. 

[2.  Prepare  a  trocar — Sivori's  pattern  (fig.  38,  p.  49)  is  very  suitable — thus: 
to  the  side  tube  attach  a  fairly  long  piece  of  india-rubber  tubing,  and  to  the 
other  end  of  the  latter  connect  a  piece  of  straight  glass  tubing  long  enough 
to  reach  from  the  mouth  to  the  bottom  of  the  flask.  Boil  the  apparatus 
for  an  hour. 

[3.  Take  the  trocar  out  of  the  water  with  a  pair  of  sterile  forceps,  pass  it 
between  the  neck  and  the  plug  of  one  of  the  sterile  flasks,  and  then  wrap 
the  plug  well  round  it.  Pass  the  glass  tube  well  down  into  the  flask  in  the 
same  way. 

[4.  Cleanse  the  skin  of  the  neck  of  the  animal,  and  pass  the  trocar  into 
the  jugular  vein  as  described  at  p.  49. 

[5.  When  the  flask  is  about  two-thirds  filled,  pinch  the  rubber  tubing,  get 
an  assistant  to  withdraw  the  glass  tubing  and  pass  it  as  in  (3)  into  a  second 
flask.  Release  the  pressure  on  the  tubing,  and  fill  the  second,  and  in  the 
same  way  the  third  and  fourth  flasks.  Care  must  be  taken  when  withdrawing 
the  trocar  in  the  first  instance,  and  the  glass  tube  later,  that  the  wool  plug 
is  so  arranged  that  no  air  channel  is  left. 

[6.  Take  the  flasks  of  blood  to  the  laboratory  and  stand  them  vertically. 
If  any  of  the  plugs  be  soiled  with  blood  or  do  not  fit  well  replace  them 
with  sterile  wool  (see  A.  2  p.  45). 

[7.  When  the  clot  has  formed  and  the  serum  separated,  proceed  as  in  A  p.  45. 

[8.  The  serum  should  be  sterile,  but  as  a  precautionary  measure  a  little 
chloroform  may  be  added  to  it  or  it  may  be  heated  to  55°-60°  C.  for  an 
hour.] 

B.  Roux  and  Nocard's  method.  Recommended. — This  method  has  the 
advantage  of  furnishing  a  much  clearer  serum  and  a  medium  more  favourable 


FIG.  37.— Nocard's  trocar. 


for  growth  than  Koch's,  and  should  therefore  be  adopted  in  preference  to  the 
latter  whenever  possible. 

Instruments  required. — 1.  A  Nocard's  trocar  (fig.  37)  on  to  the  cannula  of 


SERUM 


49 


which,  when  the  trocar  is  withdrawn,  a  metal  adjustment  can  be  fitted  ; 
this  carries  a  piece  of  red  rubber  tubing  about  50  cm.  long,  to  which  is 
attached  a  piece  of  glass  tubing  15  cm.  long  bevelled  at  its  free  end. 

The  trocar  and  the  rubber  tube  with  its  appendices  are  wrapped  separately 
in  filter  paper  and  autoclaved. 

Sivori's  trocar  (fig.  38)  may  with  advantage  be  employed  instead  of  Nocard's  ; 
it  is  provided  with  a  lateral  tube  E  to  which  the  rubber  tubing  is  attached.  The 
trocar  with  tubing  attached  can  be  sterilized  as  a  single  piece  of  apparatus,  and  the 
blood  collected  directly,  thus  avoiding  any  risk  of  contamination. 


FIG.  38.  —  Sivori's  trocar. 

M,  handle  ;   C,  shoulder  into  which  the  conical  part  AB  fits  hermetically,  so 
that  the  blood  flows  up  the  cannula  D,  and  passes  out  through  E. 

2.  A  pair  of  sterile  scissors  curved  on  the  flat  and  a  sterile  bistoury. 

3.  One  or  two  wide-mouthed  bottles  of  3  litres  capacity. 

The  bottles  are  washed  and  dried,  and  the  mouth  of  each  covered  with  two 
or  three  folds  of  paper  which  is  tied  down  round  the  neck  with  string  ;  over 
this  another  similar  but  larger  covering  is  fastened  with  string  in  a  similar 
manner,  so  that  it  can  be  removed  without  interfering  with  the  cover  beneath 
(fig.  39).  The  bottles  must  be  sterilized  in  the  hot  air  sterilizer. 

4.  Some  sterile  Chamberland  flasks  and  test-tubes. 

Technique.  —  As  a  rule,  blood  is  taken  from  an  horse  or  an  ass  while  the 
animal  is  standing.  If  necessary  its  eyes  can  be  covered  and  the  animal  can 
be  held  with  a  twitch.  If  it  is  proposed  to  bleed  a 
bovine  animal,  it  will  be  best  to  throw  it  on  a 
table  such  as  is  used  for  vaccination  inoculations.1 
The  animal,  whichever  species  is  used,  should  be 
fasting. 

1.  Proceed  as  for  bleeding  from  the  jugular  vein. 
Sterilize   the   skin.     Press  the  vein  at  the  root  of 
the  neck  to  render  it  prominent,  and  make  a  small 
longitudinal  incision  through  the  skin  with  a  bistoury 
along  the  line  of  the  vessel  on  the  distal  side  of  the 
point  of  compression. 

2.  Introduce  the  trocar  through  the  incision  and 
push   it   through  the   sub-cutaneous  tissues  for   a 
distance  of  about  2  cm.,  then  pierce  the  vein  and 
push  the  trocar  into  it  in  the  direction  of  its  long 
axis. 

3.  The  cannula  is  now  in  place  ;  withdraw  the 
trocar,  and  attach  the  metal  adjustment  carrying 
the    rubber    tube.     Meanwhile    an    assistant   com- 
presses  the  vein  above  to  prevent  blood  entering 
the  cannula.     This  must  be  done  quickly. 

4.  The  rubber  tube  being  attached,  pinch  it  firmly 

between  the  thumb  and  index  finger  of  the  left  hand.  The  assistant  releases 
the  pressure  on  the  vein  above  the  cannula,  but  maintains  the  pressure  on 
the  cardiac  side. 

I1  In  our  experience  it  has  never  been  necessary  to  throw  a  bovine  animal  :  adults 
usually  stand  quite  quietly.] 

T\ 


.  39.  -Bottle  for  collecting 


Part  of  the  outer  cover  has 
SSathm°ved  t0  Sh°W  the  One 


50 


SOLID   MEDIA 


5.  A  second  assistant  hands  the  sterile  bottle,  loosens  and  removes  the 
outer  covering,  and  perforates  the  inner  cover  with  the  glass  tube  attached 
to  the  rubber  tube.     Now  release  the  pressure  on  the  rubber  tubing,  and 
blood  will  flow  into  the  bottle. 

When  the  first  bottle  is  three-parts  filled,  stop  the  flow  of  blood  by  pinching 
the  rubber  tubing ;  the  assistant  then  withdraws  the  glass  tubing  from  the 
bottle  and  covers  the  mouth  as  quickly  as  possible  with  the  paper  cap  and 
fastens  it  round  the  neck.  Fill  a  second  bottle  in  the  same  way. 

5  to  6  litres  of  blood  can  be  taken  from  an  horse  without  harm,  but  3  litres 
is  sufficient  to  take  from  a  young  heifer. 

6.  Place  the  bottles  in  a  cool  place  for  36  hours.     The  serum,  which  is 
transparent  and  of  a  beautiful  pale  yellow  colour,  will  then  be  floating  on 
the  surface.     Decant  the  serum  from  the  clot  with  a  Chamberland  flask  [or 
Cobbett's  bulb],  being  careful  not  to  contaminate  it,  and  distribute  it  at  once 
in  sterile  tubes  as  described  above  (pp.  45  and  47). 

C.    Latapie's  apparatus.    Recommended. — The    technique    of   Eoux    and 
Nocard's  method  is  simplified  by  using  this  apparatus.     All  contamination 
is  avoided,  and  a  yield  of  about  700  c.c.  of  serum  per 
litre  of  blood  is  obtained  instead  of  400-450  c.c.  by  the 
ordinary  method. 

Description. — The  apparatus  (fig.  40)  consists  of  a 
wide-mouthed  bottle  F  capable  of  holding  several 
litres,  and  plugged  with  an  india-rubber  plug  B  per- 
forated with  three  holes.  A  number  of  glass  tubes  (t) 
open  at  both  ends  and  perforated  with  several  holes 
laterally,  is  put  into  the  bottle.  Three  pieces  of  glass 
tubing  are  passed  through  the  india-rubber  plug. 
Through  one  (a)  the  blood  enters  the  bottle,  a  piece 
of  rubber  tubing  connecting  it  with  the  cannula.  The 
tube  b  is  simply  to  allow  access  of  air  to  the  interior  ; 
it  is  plugged  above  with  wool,  while  the  other  end 
extends  some  distance  into  the  bottle  and  is  bent  in 
the  form  of  an  U.  Lastly,  the  tube  e  serves  for  the 
collection  of  the  serum  :  its  lower  end  also  bent  in  an 
U -shape  reaches  a  few  cm.  below  the  plug,  while  its 
upper  end  is  attached  by  means  of  a  piece  of  rubber 
tubing  to  a  piece  of  glass  tube  drawn  out  and  sealed 
in  the  flame.  A  clip  can  be  placed  on  the  rubber  con- 
nexion p  to  disconnect  the  two  pieces  of  glass  tubing.  Finally,  the  apparatus 
is  arranged  in  a  special  support  (not  pictured  in  the  figure)  which  allows 
the  bottle  to  be  inclined  so  that  the  neck  points  upwards  or  downwards 
at  will.1 

Technique. — 1.  Sterilize  the  apparatus  in  the  autoclave.  Moisten  the 
wool  in  the  tube  6,  wrap  the  bottle  in  filter  paper,  and  raise  the  temperature 
slowly.  After  sterilization  allow  to  cool,  and  then  lute  the  plug  with  paraffin. 

2.  Puncture  the  vein  as  in  Roux  and  Nocard's  method,  and  connect  the 
cannula  to  the  tube  a.     The  bottle  must  not  be  more  than  half-filled  and 
the  blood  must  not  reach  to  the  level  of  the  air  tube  b.    The  flow  of  blood  is 
stopped  by  clipping  the  tube  a.     (The  bottle  is  of  course  held  with  the  neck 
up  during  this  part  of  the  operation.) 

3.  Leave  for  12  hours  or  more  until  the  blood  has  clotted. 


FIG.  40. — Latapie's  ap- 
paratus, in  which  to  collect 
blood  for  serum  from  large 
animals  (horse  or  bovine). 


1  In  Chapter  XII.  an  apparatus,  designed  by  the  same  observer,  for  the  collection  of  blood 
from  small  animals  will  be  described. 


SERUM  51 

4.  When  the  clot  has  formed  and  shrunk  from  the  pieces  of  glass  tubing, 
invert  the  bottle  gently  in  its  support  so  that  the  neck  is  the  lowest  part. 
The  serum  will  then  run  down  into  the  neck. 

5.  Clip  the  tubing  at  p,  break  off  the  pointed  end  of  the  glass  tubing,  and 
plunge  it  into  the  sterile  flask  in  which  the  serum  is  to  be  collected  :  loosen 
the  clip,  and  the  serum  will  flow  out.     By  means  of  the  clip  the  rate  of  flow 
can  be  altered  at  will  or  entirely  stopped. 

The  opening  of  e  within  the  bottle  being  a  little  distance  from  the  plug,  a  small  quantity 
of  serum  containing  red  cells  remains  in  the  bottle ;  the  bend  in  the  tube  prevents  these 
cells  from  being  drawn  off  with  the  serum. 

Coagulation  of  serum. 

Serum  is  coagulated  by  heat.  In  order  that  it  may  retain  its  transparency, 
the  temperature  during  coagulation  must  not  exceed  68°-70°  C.,  and  to 
completely  solidify  the  serum  it  must  be  kept  at  this  temperature  for 
2  or  3  hours. 

The  tubes  containing  the  liquid  serum  are  sloped  as  in  the  case  of  agar. 
Coagulation  is  generally  effected  in  a  modified  form  of  the  apparatus  devised 
by  Koch  [e.g.  in  an  Hearson's  serum  coagulator.] 

[Hearson's  serum  coagulator. — In  construction  and  in  some  models  in 
appearance  Hearson's  serum  coagulator  (fig.  41)  is  the  same  as  Hearson's 


FIG.  41. — Hearson's  apparatus  for  the  coagulation  of  serum. 

warm  (37°  C.)  incubator  (p.  61),  but  in  the  former  the  capsule  is  constructed 
to  work  at  higher  temperatures  such  as  are  suitable  for  the  coagulation  of 
serum.  Special  holders  are  also  supplied  which  retain  the  tubes  in  a  slant- 
ing position.  To  maintain  a  saturated  atmosphere,  dishes  of  water  or  wet 
cloths  can  be  placed  on  the  floor  of  the  coagulator.] 

Koch's  apparatus  consists  of  a  double- walled  rectangular  copper  box  supported 
on  legs,  by  means  of  which  the  angle  which  it  subtends  with  the  horizon  can  be 
altered  at  will.  The  space  between  the  walls  is  filled  with  water,  and  the  floor  of 
the  apparatus  is  covered  with  a  thin  layer  of  sand  on  which  the  tubes  are  laid.  A 
thermometer  is  placed  alongside  the  tubes.  The  apparatus  is  closed  above  with 
a  moveable  cover  consisting  of  two  sheets  of  glass  mounted  in  a  metal  frame  with 
a  thin  layer  of  air  between  them.  The  apparatus  is  heated  by  gas,  which  passes 
through  a  Roux's  regulator  immersed  in  the  water  between  the  two  walls.  The 
technique  is  as  follows : 


52  SOLID   MEDIA 

1.  Lay  the  tubes,  each  containing  about  10  c.c.  of  serum,  in  the  sand.     Incline 
the  apparatus  so  that  the  serum  does  not  touch  the  plugs. 

2.  Light  the  gas,  and  when  the  temperature  as  shown  by  the  thermometer  inside 
reaches  68°  C.,  adjust  the  regulator  (Chap.  III.),  so  that  the  temperature  remains 
constant. 

3.  The  length  of  time  required  to  solidify  different  samples  of  serum  varies  (from 
2  to  3  hours).     A  tube  must  be  taken  out  from  time  to  time  to  ascertain  the  con- 
dition of  the  serum,  which  will  be  sufficiently  set  when  holding  the  tube  upright  it 
retains  its  slope.     [It  is  perhaps  better  to  hold  the  tube  by  the  upper  end  and  tap 
the  lower  end  firmly  against  the  thumb  nail :    if  the  serum  quiver,  the  heating  has 
not  been  continued  long  enough.]     Stop  the   heating  at  this  stage.     The  serum 
when  set  should  still  be  transparent  and  of  an  amber-yellow  colour. 

To  obtain  a  more  transparent  product,  Vagedes  advises  coagulating  in  an  atmosphere 
of  water  vapour,  and  this  can  be  effected  by  placing  Petri  dishes  filled  with  water  along- 
side the  tubes,  [or  by  placing  folded  cloths  which  have  been  wrung  out  in  warm  water 
over  the  tubes,  taking  care  that  they  do  not  touch  the  wool  plugs.] 

4.  Incubate  at  30°  [or  37°]  C.  for  36  hours,  to  ensure  their  sterility  before  using 
them  as  culture  media. 

If  only  a  few  tubes  of  serum  have  to  be  coagulated,  Koch's  apparatus  can  be  dispensed 
with,  and  the  tubes  dealt  with  as  follows.  Arrange  the  tubes  in  a  small  flat  copper  tray 
•(fig.  42),  about  12  cm.  wide,  one  side  of  which  is  notched  to  receive  the  upper  plugged 


FIG  42. — Copper  tray  on  which  to  slope  tubes  of  culture  media. 

ends  of  the  tubes,  while  the  other  ends  resting  against  the  opposite  side  keep  the  tubes 
in  a  sloped  position.  Cover  the  tray  with  a  sheet  of  glass,  stand  it  on  a  saucepan  filled 
with  water,  and  slowly  heat  the  water  to  boiling.  The  tubes  will  be  set  in  about  an 
hour  or  two. 

Other  serum  media. 

In  cases  of  simple  pleurisy  the  fluid  which  can  be  drawn  off  often  yields 
a  very  clear,  easily  coagulable  serum,  very  suitable  for  the  purposes  of  a 
culture  medium. 

To  collect  the  fluid  aseptically,  operate  in  the  usual  manner,  using  a  sterile 
Potain's  apparatus.  Boil  the  trocar,  autoclave  the  rubber  plug  and  aspirating 
tube  at  115°  C.,  and  sterilize  the  flask  [or  bottle]  in  the  hot  air  sterilizer.  The 
serum  is  distributed  into  tubes  afterwards  by  means  of  a  Chamberland 
flask  (see  also  p.  45).  An  absolutely  sterile  serum  can  frequently  be  obtained 
in  this  way,  but  it  is  nevertheless  often  necessary  to  tyndallize  the  fluid 
before  coagulating  it  (p.  47). 

Ascitic  fluid  as  a  rule  yields  only  a  poorly  coagulable  serum,  which  is  not 
of  great  value  when  a  solid  serum  is  wanted. 

Lceffler's  serum. 

1.  Prepare  a  broth  in  the  ordinary  way,  using 

Water,  -  -          -  1000  grams. 

Beef,      -  -  500 

Peptone,         -  -  20       „ 

Salt,       -  5 

Glucose,  -  10       „ 

Normal  soda  solution,  -                                         -          -  Q.S.  to  make  slightly 

alkaline. 

2.  Aspirate  1  part  of  this  broth  and  3  parts  of  sterile  liquid  serum  into  a 
Chamberland  flask. 


EGG   MEDIA  53 

[The  quantities  can,  of  course,  be  measured  out  with  sterile  pipettes  into  a 
sterile  vessel.] 
3.  Tube.     Coagulate  at  70°-75°  C. 

Glycerin-serum. 

An  excellent   culture  medium  for  the  tubercle  bacillus  is   obtained  by 
mixing  6  to  8  per  cent,  of  pure  glycerin  with  serum. 

1.  Aspirate  6  to  8  grams  of  pure  glycerin,  previously  sterilized  in  the  auto- 
clave, into  a  Chamberland  flask : 

2.  Then  100  c.c.  of  sterile  liquid  serum  into  the  same  flask.     (To  facilitate 
measurement  the  flask  can  be  graduated  beforehand.) 

3.  Tube   and  .set   at   75°  C.     This   mixture   requires  a   somewhat  higher 
temperature  than  ordinary  serum. 

Serum-agar.    Ascitic-agar. 

1.  Dissolve  1*5  grams  of  agar  in  100  c.c.  of  water.     Filter.     Tube  (about 
5  c.c.  in  each  tube).     Sterilize  at  120°  C. 

2.  Cool  to  40°  C.     To  each  tube  add  an  equal  volume  of  sterile  serum  or 
sterile  ascitic  fluid.     Mix  gently  by  rotating  the  tubes  in  the  hands.     Cool 
in  the  sloping  position. 

Blood-agar.1 

(Bezan$on  and  Griffon.) 

1.  Take  a  number  of  tubes  of  glycerin-agar,  melt  in  a  water  bath,  and  cool 
to  40°  C. 

2.  Add  to  each  tube  a  small  quantity  (about  1  c.c.)  of  blood  from  a  rabbit's 
artery  (Chap.  XII. ).     Mix  without  shaking  the  tubes,  and  cool  in  a  sloping 
position. 

A  solution  of  haemoglobin  (p.  34)  may  be  used  instead  of  blood  in  this 
case. 

Serum-agar. 

(Tochtermann.) 

1.  Dissolve  in  500  c.c.  of  boiling  water 

Peptone  (Chapoteaut,  or  Witte), 5  grams. 

Salt,       -  2-50   „ 

Glucose,  2-50   „ 

Chopped  and  washed  agar,        -  -         -         -         -         -         10        „ 

2.  Mix  with  the  above  solution  500  c.c.  of  sheep  serum,  and  autoclave  for 
30  minutes  at  115°-120°  C. 

3.  Filter   in   the   warm   through   moistened   Chardin   paper.     Tube   and 
sterilize  at  115°  C. 

Egg. 
Eggs  can  be  used  for  the  cultivation  of  micro-organisms  in  several  ways. 

A.  Take  a  fresh  egg,  shake  it  vigorously  to  mix  the  white  and  the  yolk  : 
wash  the  shell  in  perchloride  of  mercury  and  dry  with  sterile  filter  paper. 
Flame  the  narrow  end  until  the  shell  blackens.     Make  a  hole  with  a  sterile 
metal  point.     Pass  a  platinum  wire  or  pipette  charged  with  the  material  to 
be  sown  through  the  hole,  then  close  the  latter  with  a  little  melted  Golaz's 
wax.     It  is  well  to  coat  the  egg  with  a  layer  of  collodion. 

B.  Take  a  fresh  egg,  flame  the  pointed  end,  make  a  hole  as  described  above  : 
aspirate  the  white  into  a  sterile  pipette.     Tube  in  sterile  tubes.     Coagulate 

at  70°  C.  as  in  the  case  of  serum. 

« 
1  See  also  under  Pfeiffer's  bacillus  and  Gonococcus. 


54  SOLID   MEDIA 

C.  Boil  an  egg  hard.     Remove  the  shell,  cut  up  the  egg  into  pieces,  and 
place  in  small  Petri,  or  other  covered  glass,  dishes.     Sterilize  the  watch-glasses 
or  dishes  at  115°  C. 

D.  Lubenau  recommends   yolk  of   egg  media  for  growing  tubercle  and 
diphtheria  bacilli.     The  procedure  is  as  follows  : 

1.  Prepare  a  neutral  broth  (p.  30)  containing  1  per  cent,  of  glucose  (for 
the  diphtheria  bacillus)  or  3  per  cent,  of  glycerin  (for  the  tubercle  bacillus). 
Distribute  in  quantities  of  100  c.c.  in  2J-litre  flasks.     Sterilize. 

2.  Wash  an  egg  with  warm  water  and  soap.     Lay  the  egg  in  a  Petri  dish, 
pour  a  little  alcohol  over  it,  and  set  light  to  the  alcohol. 

Make  a  hole  in  the  shell  with  a  sterile  instrument,  and  pour  the  yolk  into 
one  of  the  flasks  of  broth.  Add  the  yolks  of  five  eggs  to  each  flask.  Shake 
the  flasks  well. 

3.  Distribute    the    medium    into    tubes.     Slope   the    tubes    in    a    serum 
coagulator  (p.  51).      Heat  for  2  or  3  hours  at  90°  C.  on  three  successive 
days. 

[E.  Dorset's  egg  medium. — "  The  eggs  are  thoroughly  cleansed  with  water 
of  any  adherent  dirt,  and  then  washed  with  5  per  cent,  carbolic  solution, 
and  allowed  to  partially  dry.  The  ends  of  the  eggs  are  then  gently  dried  in 
the  flame,  and  pierced  with  a  burned  sharp  forceps.  The  hole  at  one  end 
should  be  about  f  in.  in  diameter,  and  the  membrane  broken  ;  the  other 
end  which  is  to  be  blown  into  should  be  smaller,  and  the  membrane  left 
unbroken  if  possible.  The  eggs  are  then  blown  into  a  sterile  Erlenmeyer 
flask,  the  blowing  being  done  from  the  cheeks,  which  will  help  to  avoid 
spilling  saliva  and  leakage  of  air  around  the  outside  of  the  egg.  To  the  egg 
is  then  added  10  per  cent,  of  water  by  volume  of  the  weight  of  the  eggs. 
The  mixing  is  done  by  a  twirling  motion  of  the  flask  or  by  gently  stirring  with 
a  glass  rod.  Bubbling  is  to  be  sedulously  avoided.  The  mixture  is  then 
strained  through  cheese-cloth  by  gravity  and  tubed.  The  tubes  are  then 
inspissated  at  70°  C.  for  2-2  J  hours  in  a  moist  chamber  "  (Park  and  Krum- 
weide).] 

Meat. 

Into  a  litre  flask  put  500-600  grams  of  finely  chopped  lean  beef.  Add 
sufficient  normal  soda  solution  to  make  the  reaction  neutral  or  slightly 
alkaline.  Plug  the  flask  with  wool.  Sterilize  at  115°  C. 

Internal  organs. 

The  placenta,  liver,  spleen,  kidneys,  etc.,  can  be  used  as  culture  media. 
The  organs  must  be  removed  with  the  usual  aseptic  precautions  from  healthy 
animals  which  have  been  recently  killed. 

The  technique  recommended  by  Gueniot  for  the  preparation  of  placenta 
will  serve  as  an  example  of  the  method  of  preparing  these  culture 
media. 

1.  Lay  the  placenta  (if  possible  receive  it)  in  a  sterile  basin  with  the 
uterine  surface  uppermost.     Scorch  this  surface  with  a  large  heated  metal 
plate. 

2.  Cut  off  a  number  of  pieces  with  a  sterile  forceps  and  scalpel,  and  place 
them  with  the  scorched  surface  downwards  in  sterile  Petri  dishes,  or  better 
in  large  sterile  tubes. 

Place  the  dishes  and  tubes  in  the  incubator  at  37°  C.  for  a  day  or  two  to 
control  the  technique.  Thos£  that  remain  sterile  (at  least  60  per  cent.)  can 
then  be  used  as  culture  media. 


VEGETABLE   MEDIA  55 

4.  Vegetable  media. 

Potato. 

A.  Petri  dish  method.— 1.  Select  a  number  of  perfectly  sound  potatoes,  scrub 
away  the  soil  adhering  to  them  in  running  water,  then  dry  and  peel  them. 

2.  Cut  them  into  slices  about  10-15  mm.  thick  parallel  to  their  long  axes, 
and  drop  the  slices  into  a  dish  of  distilled  water. 

The  slices  should  not  be  touched  with  the  fingers,  and  it  is  best  to  use  a 
silver  blade  as  steel  often  turns  potatoes  black. 

3.  Dry  the  pieces  between  folds  of  white  filter 
paper. 

4.  Then  lay  them  in  Petri  dishes  (fig.  43)  or  other 
suitable  covered  glass  dish. 

5.  Sterilize  the  potato  in  the  dishes  at  120°  C.  FIG  43  —Petri  dish 
for  20  to  30  minutes. 

Potatoes  must  be  sterilized  at  120°  C.,  because  a  highly  resistant  organism 
(the  potato  bacillus),  which  is  often  present  on  the  surface,  may  in  slicing  the 
potatoes  be  carried  by  the  knife  on  to  the  cut  surface. 

B.  Method  recommended. — 1.  Wash  and  scrub  the  potatoes  as  above. 

2.  Cut  the  potatoes,  not  into  slices,  but  into  elongated  parallelepipeds  or 
semi-cylindrical  pieces  4  to  5  cm.  long,  so  that  they  can  be  put  in  special 

potato-tubes  also  known  as  Roux's  tubes. 
CQ^)  These  tubes  (fig.  44)  are  rather  wider  than  ordinary  culture- tubes  and 

the  potato  rests  on  a  constriction  situated  about  the  lower  one- fourth ; 

the  bulb  below  collects  the  condensation  water. 

A  special  cutter  may  conveniently  be  used  for  slicing  the  potatoes, 

but  the  only  advantage  to  be  gained  is  that  the  pieces  are  more  neatly 

and  regularly  cut.     The  slices  should  not  be  too  long,  otherwise  they 

will  curl  when  boiled. 

3.  Wash  the  pieces  in   distilled  water  :    dry  between  blotting 
paper. 

4.  Put  them  into  tubes.     Plug  with  wool. 

5.  Sterilize  as  above. 

Note. — Potatoes,  though  generally  neutral  in  reaction,  are  sometimes 
strongly  acid,  in  which  case  they  are  not  suitable  for  the  cultivation  of 
bacteria.     If  it  be  necessary  to  use  these  acid  potatoes,  they  must  be 
soaked  for  some  hours  before  being  sterilized  in  a  0'5  per  cent,  solution 
FIG.  44. —  of  soda. 
Potato-tube. 

[C.  Glycerin-potato.— 1.  After  cutting  the  potatoes  into  suitably 
shaped  pieces  as  above  (B),  soak  them  in  a  dilute  (1-1000)  solution  of  sodium 
carbonate  for  24  hours. 

[2.  Transfer  the  pieces  to  a  5  per  cent,  solution  of  glycerin  in  water  for  a 
further  24  hours. 

[3.  Tube  in  ordinary  test-tubes,  which  should  have  a  pledget  of  wool  at  the 
bottom.  Fill  up  the  tubes  with  the  5  per  cent  glycerin  solution. 

[4.  Sterilize  at  100°  C.  on  three  successive  days. 

[5.  When  required  for  use  pour  off  nearly  all  the  glycerin  solution,  and  sow 
the  surface  of  the  medium.] 

D.  Potato  mash.— 1.  Peel  the  potatoes,  cut  them  into  large  pieces,  and 
boil  them  in  water. 

2.  Pass  them  through  a  sieve. 

3.  Distribute  the  mash  in  layers  1-2  cm.  thick  in  Petri,  or  other  covered 
glass,  dishes. 

4.  Sterilize  at  120°  C.  for  20  minutes. 


56 


SOLID   MEDIA 


Starch  jelly. 

To  180  grams  of  water  add  10  grams  of  potato  meal  and  5  grams  pre- 
cipitated calcium  carbonate.  Distribute  in  Erlenmeyer  flasks  or  Petri 
dishes  and  sterilize  at  115°  C.  When  the  starch  cools  it  forms  an  homogeneous 
whitish  layer  on  the  bottom  of  the  vessel. 

Heinemann's  jelly. — Heinemann  recommends  the  following  jelly  in  place  of 
potato.  The  artificial  medium  has  the  advantage  of  being  of  constant  composition 
and  reaction. 

1.  Prepare  the  following  solution  : 

5  grams. 

2 

2 


Asparagin, 

Di-potassium  phosphate, 
Di-sodium  phosphate, 
Magnesium  sulphate, 
Calcium  chloride,     - 
Ammonium  lactate, 
Water,  - 


2 

2 

2 

200 


2.  Dissolve  15  grams  of  agar  and  10  grams  of  peptone  in  the  warm  in  600  grams 
of  water. 

3.  Mix  the  two  solutions.     Make  neutral  to  phenol-phthalein.     Filter. 

4.  After  filtering,  and  while  still  hot,  add  30  grams  of  starch  made  into  an  homo- 
geneous suspension  with  a  little  water.     Boil  the  mixture  for  several  minutes. 

5.  Distribute  in  tubes.      Sterilize  at  120°  C.  for  5  minutes.      Slope  the  tubes 
and  allow  them  to  cool. 

Bread. 

A.  Soak  some  slices  of  white  bread  in  distilled  water,  place  them  in  covered 
glass  dishes,  and  sterilize  at  115°  C.  for  20  minutes. 

B.  1.  Crumble  some  bread  and  dry  it  in  the  air  between  sheets  of  filter 
paper. 

2.  When  dry,  grind  it  up  in  a  coffee  mill. 

3.  Put  the  powder  in  layers  1-2  cm.  thick  in  Petri  dishes  or  in  Erlenmeyer 
flasks,  and  add  sufficient  distilled  water  to  soak  all  the  bread  (about  2J  parts 
of  water  to  1  part  of  bread  by  weight). 

4.  Sterilize  at  115°  C.  for  20  minutes. 

Rice  milk. 

1.  Mix  intimately 

Milk,      -          -  150  grams. 

Peptone  broth,        -  50         ,, 

Powdered  rice,         -         -  -       100         ,, 

2.  Distribute  the  mixture  in  Petri  dishes  in  layers  1-2  cm.  deep. 

3.  Heat  to  115°  C.  for  20  minutes.     The  mixture  solidifies  and  forms  an 
opaque  white  layer. 

5.  Coloured  media. 

Coloured  media  are  used  for  the  recognition  of  particular  micro-organisms 
which  produce  changes  of  colour  in  them.  Only  a  few  formulae  are  given  here, 
the  majority  being  reserved  for  description  later  (vide  the  typhoid  bacillus, 
the  colon  bacillus,  etc.). 

Media  tinted  with  blue  litmus  [or  neutral-red]  and  containing  a  carbo- 
hydrate are  the  most  generally  used  :  organisms  which  ferment  carbohydrates 
with  formation  of  acid,  when  grown  in  a  litmus  medium  change  the  colour 
of  the  litmus  from  blue  to  red  [and  in  a  neutral  red-medium  produce  a  bright 
red  colour], 

Preparation  of  litmus  solution. — Granulated  litmus  is  ground  up,  85  per  cent, 
alcohol  is  poured  on  to  the  powder,  and  the  mixture  boiled.  6  to  8  parts  of 
water  are  added  to  the  residue,  and  the  resulting  liquid  mixture  is  heated  and 


COLOURED   MEDIA  57 

filtered  through  paper.  The  filtrate  is  kept  in  a  flask  plugged  with  wool.  To  one- 
half  of  this  liquid  sulphuric  acid  is  added  until  the  colour  is  nearly  red,  and  the 
other  half  then  added  to  it ;  a  sensitive  indicator  is  thus  obtained.  The  solution 
is  distributed  in  tubes  which  are  plugged  and  sterilized  at  115°  C. 

Litmus-lactose-gelatin. 

1.  Prepare  and  sterilize  a  number  of  tubes  of  gelatin  in  the  ordinary  way 
(p.  39),  adding  (at  stage  4)  2  to  4  per  cent,  of  lactose. 

2.  Prepare  a  number  of  tubes  of  sterilized  litmus  solution. 

3.  Just  before  use,  melt  the  lactose-gelatin  in  a  water  bath  and  to  each 
tube  add  with  a  sterile  pipette  sufficient  sterile  litmus  solution  to  impart  a 
distinctly  blue  colour  to  the  medium. 

Never  sterilize  a  medium  after  adding  litmus  :    the  subsequent  heating  is  liable 
to  discharge  the  blue  colour. 

Litmus-glucose-gelatin,  litmus-mannite-gelatin,  etc.,  and  various  litinus- 
agars  are  prepared  in  a  similar  manner. 

Barsiekow's  medium. 

Prepare  separately  the  two  following  solutions  and  sterilize  them : 

A.  Sodium  chloride,      ......       0*5  gram. 

Nutrose,          -------       1         „ 

Water,  -  -         -     75  grams. 

B.  The  carbohydrate  (lactose,  mannite,  etc.),  -         -       I  gram. 
Water,  -  ......     25  grams. 

Litmus  solution,      ......       Q.S.  to  give  an  amethyst  tint 

to  the  solution. 

After  cooling,  mix  the  two  solutions  and  distribute  in  tubes. 

Litmus  milk. 

Add  a  sufficient  quantity  of  sterile  litmus  solution  to  sterile  milk.  [It  is 
highly  important  that  the  reaction  of  the  milk  should  be  neutral.  Milk 
bought  in  shops  is  often  acid,  in  which  case  a  sufficiency  of  sodium  carbonate 
must  be  added  to  neutralize  the  medium.] 

Noeggerath's  medium. 

1.  Mix  in  the  following  proportions  saturated  aqueous  solutions  of  the  dyes 
mentioned  : 

Methyl-blue,  -  2  c.c. 

Gentian-violet,         ...  4     „ 

Methyl-green,  1     „ 

Chrysoidine,  -  4     „ 
Fuchsin,                    ........           3,, 

2.  Add  200  c.c.  distilled  water. 

3.  The  mixture  now  has  a  neutral  greyish- blue  tint :    leave  it  to  stand  for  a 
fortnight,  then  if  the  colour  has  altered  bring  it  back  to  the  neutral  tint  by  the 
addition  of  any  colour  that  is  required.     Sterilize  at  100°  C. 

4.  Immediately  before  use  add  7  to  10  drops  of  the  sterile  mixture  to  a  tube  of 
agar  or  gelatin  previously  melted  in  the  water  bath. 

In  place  of  the  above  mixture,  Gasser  prefers  to  add  20  drops  of  a  saturated 
aqueous  solution  of  fuchsin  to  each  tube  of  melted  agar. 

These  media  were  recommended  by  their  authors  for  the  diagnosis  of  the  typhoid 
bacillus,  but  have  now  fallen  into  disuse  (vide  the  typhoid  bacillus). 


CHAPTER   III. 
INCUBATORS. 

Introduction. 

Section  I. — Devices  for  automatically  regulating  the  temperature  of  incubators,  p.  59. 

Section  II. — Incubators  heated  by  coal  gas,  p.  61. 

Section  III. — Incubators  heated  by  electricity,  p.  65. 

Section  IV. — Incubators  heated  by  petrol,  gasoline,  or  petroleum  oil,  p.  66. 

AN  incubator  is  a  piece  of  apparatus  designed  to  maintain  cultures  of 
organisms  constantly  at  any  temperature  which  may  be  best  suited  to  their 
growth. 

The  shape  of  the  incubator,  provided  it  be  adapted  to  the  size  of  the  tubes, 
flasks,  etc.,  which  it  will  have  to  receive,  is  generally  speaking  of  little  conse- 
quence, though  on  the  whole  the  rectangular  form  is  the  most  convenient 
because  the  space  can  be  most  completely  utilized. 

Any  metal  box  which  had  a  door  and  could  be  heated  by  a  convenient  source  of 
heat  could  in  an  emergency  be  made  to  serve  as  an  incubator.  A  rectangular  tin  or 
copper  box  for  instance,  raised  on  a  suitable  stand,  and  heated  by  a  [Bunsen  burner 
or]  small  oil  lamp,  placed  below  it  at  such  a  distance  as  to  keep  the  temperature 
within  the  box  at  the  level  required,  would  do  quite  well.  But  with  such  a  rough 
and  ready  piece  of  apparatus  the  difficulty  would  be  to  keep  the  temperature  con- 
stant, for  quite  apart  from  the  question  of  the  control  of  the  heat  supply  the 
temperature  would  be  influenced  by  the  temperature  of  the  outside  air.  And  in 
practice  these  difficulties  are  so  considerable  that  it  has  been  found  necessary  to 
design  special  forms  of  apparatus  in  which  the  temperature  can  be  kept  more  fully 
under  control.  These  of  course  are  more  complicated  and  more  expensive  than 
the  simple  arrangement  just  referred  to,  but  are  nevertheless  indispensable  if 
satisfactory  results  are  to  be  obtained. 

There  are  two  essential  points  for  which  provision  must  be  made  in  the 
construction  of  an  incubator. 

Firstly,  the  instrument  must  be  protected  as  far  as  possible  from  variations 
in  the  atmospheric  temperature,  and  from  loss  of  heat  by  radiation  and  con- 
vection. 

Secondly,  it  must  have  some  form  of  automatically  acting  regulator,  which  will 
readily  respond  to  variations  of  temperature. 

The  former  condition  is  satisfied  by  surrounding  the  outer  surface  with 
some  non-conductor  of  heat,  e.g.  wood  or  felt  or  a  water  jacket ;  or,  since 
polished  metal  surfaces  radiate  heat  very  feebly,  a  veneer  of  brightly  polished 
copper  will  serve  the  purpose  equally  well. 

To  get  the  most  satisfactory  results,  the  temperature  throughout  the 
incubator  must  be  as  uniform  as  possible.  If  the  incubator  were  an  hermeti- 


TEMPERATURE   REGULATORS 


59 


cally  sealed  box,  there  would  be  very  marked  differences  of  temperature  at 
different  levels,  and  the  larger  the  incubator  the  more  noticeable  would 
this  be.  [In  very  large  incubators,  such  as  incubator  rooms,  it  is  even 
necessary  to  have  thermometers  on  each  shelf  and  at  different  places  on  each 
shelf,  because  the  differences  of  temperature  in  different  parts  of  the  room 
are  so  considerable.]  Hence  to  maintain  as  uniform  a  temperature  as  possible 
in  an  incubator  of  the  ordinary  size  the  shelves  are  perforated  with  holes 
to  allow  of  free  circulation  of  the  air,  and  in  some  forms  ventilation  holes  are 
provided  in  the  floor  and  roof.  [With  incubators  surrounded  by  a  water 
jacket  however  this  is  not  necessary.] 


SECTION  I.— DEVICES  FOR  AUTOMATICALLY  REGULATING  THE 
TEMPERATURE   OF  INCUBATORS. 

Various  ingenious  pieces  of  mechanism  have  been  devised  for  the  purpose 
of  automatically  regulating  the  temperature  of  incubators.  Some  of  these 
regulators  are  intended  to  be  used  when  coal  gas  is  the  source  of  heat,  others 
are  constructed  for  use  with  paraffin  oil,  etc.,  but  the  former  are  the 
most  satisfactory,  and  are  the  only  ones  that  can  be  recom- 
mended.1 Those  that  are  in  most  general  use  are  described 
below. 

A.  Electric  regulators. — Of  these  Babes'  may  be  mentioned 
as  a  type.     They  are  very  complicated,  uncertain  in  action, 
and  have  no  advantage  over  the  following. 

B.  Mercury  regulators. — To  explain  the  principle  of  the 
mercury  regulators,  Chancel's  may  be   described.     The   gas 
enters  through  the  glass  tube  A  (fig.  45),  which  terminates 
within  the  regulator  in  an  oblique  opening  through  which 
the  gas  issues  and  passes  to  the  burner  through  the  side  tube  B. 
When  the  regulator  is  placed  within  the  incubator,  the  mer- 
cury contained  in  the  lower  part  R  expands  as  the  temperature 
rises,  so  that  in  time  it  obliterates  the  oblique  opening  of  the 
tube  A,  and  consequently  diminishes  the  volume  of  gas  passing 
to  the  burner  (in  the  vertical  limb  of  A  there  is  a  small  safety 
opening  0  which  permits  of  a  very  small  supply  of  gas  to 
the  burner,  just   sufficient  to  prevent  the  light  going  out 
altogether  when  the  opening  below  is  completely  obstructed 
by  the  mercury). 

When  the  temperature  within  the  incubator  drops,  the  level   FlG  45._Mercury 
of  the  mercury  falls  and  the  supply  of  gas  to  the  burner  is         regulator, 
increased.     The  regulator  is  controlled  by  the  screw  V :  when 
this  is  turned  clockwise  the  level  of  the  mercury  E  stands  at  a  higher  level  for 
any  given  temperature,  while  by  turning  it  contra-clockwise,  the  volume  of 
mercury  in  the  tube  is  diminished.     This  regulator  is  cheap  but  not  very 
sensitive,  and  is  only  correct  within  about  3°  C. ;    an  improved  form  of  it 
has  been  devised  by  Arloing. 

C.  Ether  regulators.— Rohrbeck's  (fig.  46)  may  be  taken  as  an  example  of 
this  form  of  regulator,  the  working  of  which  depends  upon  alterations  in  the 
vapour  tension  of  ether  at  different  temperatures. 

1  To  ensure  as  constant  a  temperature  as  possible  with  gas,  a  pressure-regulator  should 
be  affixed  to  the  main,  so  that  the  gas  always  reaches  the  incubator  at  a  constant 
pressure. 


60 


INCUBATORS 


Entering  the  smaller  tube  A  (fig.  46),  the  gas  passes  by  way  of  its  lower 
obliquely  cut  end  through  B  to  the  burner.  The  outer  tube  is  divided 
towards  its  lower  part  by  means  of  a  funnel-shaped  glass  partition  E  into  an 
upper  and  lower  part.  The  lower  part  R  is  filled  below  with  mercury,  and 
above  with  ether  vapour.  When  the  surrounding  temperature  rises  the 
pressure  of  ether  vapour  increases,  and  the  mercury  rises  in  the  funnel  and 
gradually  more  or  less  occludes  the  lower  opening  of  A,  thus  cutting  off  the 
supply  of  gas  to  the  burner.  A  pilot  opening  0  is  provided,  so  that  the  gas 
shall  not  be  completely  extinguished.  The  apparatus  is  regulated  by  raising 
or  lowering  the  tube  A.  The  regulator  is  sensitive  but  fragile. 


FIG.  46. — -Ether  regulator. 


FIG.  47.— Air  regulator. 


D.  Air  regulators.— Of  these,  Bohr's  may  be  taken  as  an  example  (fig.  47). 
In  principle  they  are  similar  to  the  ether  regulators,  air  replacing  the  ether 
in  the  latter. 

The  regulator  is  fitted  to  the  incubator  with  the  tap  R  open,  and  the 
reservoir  A  full  of  air.  When  the  temperature  of  the  incubator  has  reached 
the  temperature  required,  the  tap  R  is  closed.  If  now  the  temperature  of 
the  incubator  be  raised  the  air  expands,  presses  on  the  mercury  contained 
in  the  U  -tube  B  and  forces  it  upwards  thus  partially  or  completely  occluding 
the  oblique  opening  C  of  the  gas  supply  tube,  and  so  cutting  off  more  or  less 
completely  the  gas  passing  to  the  burner  through  D.  The  gas  delivery  tube 
is  provided  with  a  safety  opening  0  as  usual.  The  regulator  is  sensitive, 
but  being  affected  by  alterations  in  the  atmospheric  pressure  requires  a 
certain  amount  of  supervision. 

E.  Roux's  metal  regulator. — This  regulator  is  composed  entirely  of  metal 
(fig.  48).     It  consists  of  a  strip  of  zinc  and  a  strip  of  steel  soldered  together, 
and  then  bent  in  the  form  of  an  U.     The  metal  with  the  greater  co-efficient 
of  expansion,  zinc,  being  on  the  outside,  it  follows  that  any  increase  of  tem- 
perature will  cause  the  open  ends  of  the  U  to  approach  each  other ;    and 
conversely  if  the  temperature  of  the  metals  be  lowered  the  gap  between  the 
limbs  is  widened. 


TEMPERATURE  REGULATORS 


61 


FIG.  48. — Roux's  metal  regulator. 


The  left  limb  of  the  U  is  fixed  while  the  right  limb  R  is  free,  and  therefore 

any  change  of  shape  resulting  from  a  rise  or  fall  of  temperature  in  the  incu- 
bator is  integrated  on  the  free  limb  R,  and  transmitted  by  means  of  a  rigid 

horizontal  bar  T  to  a  piston  placed 

outside  the  incubator  which  controls 

the  supply  of  gas. 

The  tube   C   being  connected  to  a 

gas  tap,  the  gas  must  pass  under  E  to 

reach  the  chamber  to  which  the  tube 

D  leading  to  the  burner  is  connected. 
As  the  temperature  in  the  incubator 

rises,  the  free  limb  R  is  drawn  towards 

the  other  limb  taking  the  rigid  bar  T 

with  it ;    the  piston,   controlled  by  a 

spring,   closes    (this  is  shown  in  'the 

figure),  only  leaving  a  small  safety  hole 

or  by-pass  V  by  which  the   gas   can 

pass  to  the  burners,  and  accordingly 

the   temperature    is    lowered.     When 

the  temperature  is  too  low,  the  changes 

described  are  reversed ;  the  bar  is  pressed  upon  by  the  right  limb  of  the  U, 

and  this  in  turn  forces  the  piston  E  out  so  that  more  gas  passes  to  the  burners 

and  the  flames  are  larger.     After  a  few  oscillations,   the  temperature  in 

the  incubator  will  become  constant. 

By  altering  the  length  of  the  rigid   bar  T  by  means   of   a   screw,   the 

temperature  can  be  raised  or  lowered  as  required ;    or  as  in  the  pattern 

shown  in  the  figure,  which  gives  more  delicate  control,  the  length  of  the 

rigid  bar  is  fixed  while  the  length  of  the  piston  can  be  altered  by  means  of 

screws. 

In  some  cases  the  zinc  and  iron  bars  are  straight,  and  the  apparatus  assumes 

the  form  of  a  metal  tube.      This  modification  is  useful  when  the  regulator 

has  to  be  immersed  in  water,  as  for  instance 
in  a  water  bath  or  a  water- jacketed  in- 
cubator. 

Roux's  regulator  can  be  utilized  for  con- 
trolling the  temperature  of  gas  stoves  used 
for  heating  incubating  rooms  and  stoves  in 
laboratories. 

SECTION    II.— INCUBATORS   HEATED   BY 
COAL   GAS. 

A.  Hearson's  incubators  (fig.  49).— [These 
are  most  satisfactory  incubators,  and  are 
almost  if  not  quite  the  only  ones  used  in  this 
country.  There  are  two  forms,  a  "  hot  in- 
cubator "  for  temperatures  of  about  37°  C. 
and  a  "  cool  incubator  "  for  20°  C.  or  there- 
abouts. Incubators  on  the  same  principle 
FIG.  49.— Hearson's"  warm  ''incubator  can  however  be  obtained  to  work  at  any 

arranged  to  work  with  gas.  , 

temperature  above  16  C. 

[1.  The  "warm"  incubator. —This,  when  once  set,  will  work  perfectly  for 
months  together  without  adjustment  of  any  part  and  without  any  attention 
beyond  the  occasional  addition  of  a  little  water  to  replace  the  small  amount 


62 


INCUBATORS 


lost  by  evaporation.  In  actual  practice  it  is  found  that  the  temperature  can 
be  maintained  within  half-a-degree  centigrade  in  spite  of  great  changes  of 
gas  pressure  and  of  air  temperature  in  the  room  in  which  the  incubator  is 
working. 

[The  incubator  is  rectangular  in  shape,  and  consists  of  a  chamber  sur- 
rounded on  five  sides  by  a  stout  copper  water  jacket  enclosed  in  an  outer 
wooden  case  with  panels  of  uralite,  the  space  between  the  case  and  the  water 
jacket  being  packed  with  some  non-conducting  material.  The  sixth  side  is 
closed  by  a  double  door,  the  inner  of  glass,  the  outer  of  wood  also  panelled 
with  uralite. 

[The  heat  is  supplied  below  by  an  ordinary  fish-tail  gas  burner,  the  supply 
of  gas  necessary  to  maintain  the  temperature  being  controlled  by  a  capsule 
let  in  to  the  roof  of  the  incubator.] 

The  regulator  is  based  upon  the  same  principle  as  that  of  Rohrbeck  (p.  59). 
An  hermetically  sealed  metal  capsule  containing  a  few  drops  of  a  liquid 
boiling  at  the  temperature  at  which  the  apparatus  is  required  to  work  is 
placed  within  the  incubator.  When  the  liquid  in  the  capsule  boils,  the 
expansion  of  the  liquid  lifts  the  upper  part  of  the  capsule,  and  so  raises  a 
metal  rod  which  actuates  a  lever  controlling  the  supply  of  gas.  A  small 
safety  tube  prevents  the  gas  being  extinguished  when  the  main  supply  is 
cut  off  by  the  lever. 

[Technique. — As  full  instructions  are  attached  to  every  incubator,  it  is 
unnecessary  here  to  describe  the  details.] 

[2.  Hearson's  "  cool "  incubator. — The  incubating  chamber  is  similar  in 
construction  to  the  "  hot  "  incubator,  but  on  the  top  is  a  metal  box  of  the 
WATER  same  size  as  the   incubator,   surrounded   by   a 

thick  layer  of  non-conducting  material  or  wood, 
and  with  a  large  hole  in  the  top  through  which 
ice  can  be  introduced.  The  temperature  is  con- 
trolled by  a  capsule  similar  to  that  used  for  the 
hot  incubator,  but  designed  to  work  at  about 
20°  C. 

[The  source  of  heat  is  a  small  bath  of  water 
placed  at  the  side  but  near  the  top  of  the  in- 
cubator, and  heated  by  a  Bunsen  burner.  The 
capsule  controls  a  small  pipe  connected  with  a 
supply  of  water  in  such  a  way  that  when  the 
temperature  is  that  for  which  the  capsule  is  set, 
the  water  runs  to  waste ;  when  the  temperature 
has  fallen  below  that  required  the  pipe  conveys 
the  water  to  the  little  bath,  and  hot  water  runs 
into  the  incubator  jacket,  displacing  some  of  the 
cooler  water.  When  the  temperature  is  too  high 
the  cold  water  runs  directly  into  the  water 
insufficient  to  reduce  the  temperature  then  ice 
In  this  country  it  is  very  rarely,  if  ever,  that  ice 


FIG.  50. — Hearson's  "  cool 
incubator. 


jacket,  and   if   this   be 
must  be  put  in  the  box. 
is  required. 

[The  only  difficulty  likely  to  be  experienced  with  this  incubator  is  in  con- 
nexion with  its  water  supply.  If  the  supply  be  taken  directly  from  the  main, 
the  water  may  be  cut  off  or  the  pressure  for  one  reason  or  another  be  so 
reduced  from  time  to  time  that  the  automatic  regulation  breaks  down  ;  so 
that  for  satisfactory  working  it  is  desirable  to  have  a  tank  from  which  a 
supply  of  water  under  constant  pressure  may  be  derived.] 


INCUBATORS   HEATED   BY   COAL  GAS 


63 


FIG.  51. — Babes'  incubator,  fitted  with  a  Chancel  regulator. 

B.  Babes'  incubator. — One   of  the  simplest   forms  of  incubator   is   that 
known    as    Babes'.      It   consists   of  a   metal   box   covered   with    a    layer 
of  felt,  and  heated  by  a  gas  flame  the  height 

of  which  is  controlled  by  one  or  other  of 
the  many  forms  of  regulators  devised  for  the 
purpose  (p.  59). 

C.  D' Arson val's  incubator. — The  control  of  the 
temperature    in    d' Arson  val's    incubator   depends 
upon  the  changes  of  shape  which  an  elastic  lamina 
undergoes  under  the  influence  of  changes  of  pres- 
sure. 

The  incubator  is  surrounded  by  a  water  jacket 
(2,  fig.  52),  of  which  the  outer  wall  is  closed  below 
by  a  flexible  sheet  of  steel,  (3,  fig.  52),  which  also 
forms  the  roof  of  a  chamber,  10,  into  which  a  tube, 
12,  connected  with  the  gas  supply  passes.  From 
this  chamber  the  gas  reaches  the  burners  through 
two  tubes,  13  and  13'.  The  end  of  the  tube,  12, 
can  be  made  to  approach  or  recede  from  the  steel 
lamina,  3,  by  means  of  a  screw.  When  the  tube 
touches  the  steel  the  gas  is  completely  cut  off, 
and  conversely,  when  they  are  separated  the  gas 
can  pass  freely  to  the  burners. 

The  space  between  the  two  walls  is  hermetically 
sealed  everywhere,  except  above  where  an  opening, 
5,  is  left  for  the  purpose  of  filling  the  space  with 
water.  Suppose  it  be  required  to  regulate  the 
apparatus  for  a  temperature  of  37°  C.  The  tube, 
12,  is  screwed  down  away  from  the  steel  lamina, 
3,  so  that  the  gas  burns  with  a  full  flame.  When 

the  thermometer  in  the  incubator  registers  36°  C.,     FlG  53.— D'Areonyal's  incubator 
the  tube  is  raised  towards  the  lamina  so  that  .the  section. 


64 


INCUBATORS 


size^of  the  flames  is  somewhat  diminished.  If  now  the  opening,  5,  be  hermetically 
plugged,  any  further  increase  of  temperature  causes  the  water  to  expand  within 
the  double  wall,  and  so  to  force  the  lamina  downwards,  with  the  result  that  the 
gas  is  cut  off.  In  practice,  instead  of  completely  sealing  the  opening  a  plug 
carrying  a  piece  of  glass  tubing  is  inserted,  and  as  the  water  expands  it  rises  in 
this  tube  and  at  the  same  time  the  pressure  on  the  bottom  of  the  wall  of  the 
incubator  increases,  and  the  lamina  is  pressed  down. 

Note. — Recently-boiled  water  should  be  used  for  re-filling  the  incubator  :  if  tap  water 
be  used  the  bubbles  of  air  which  are  driven  off  when  the  water  is  heated  will  alter  the 
water  level,  and  the  mechanism  will  be  disturbed. 


FIG.  53. — Roux's  incubator. 

In  another  form  the  regulator  is  placed  in  the  side  of  the  incubator,  and  a  rubber 
membrane  which  is  more  sensitive  is  substituted  for  the  steel  lamina. 
D'  Arson val's  incubator  has  several  disadvantages. 

1.  By  reason  of  its  shape  only  a  small  part  of  its  total  capacity  is  available 
for  use. 

2.  The  regulation  of  the  temperature  being  a  function  of  the  level  of  the  water 
in  the  tube,  8,  it  follows  that  the  temperature  within  the  incubator  rises  as  the 
height  of  the  water  falls.     Since  this  may  happen  as  a  result  of  evaporation,  leaking 


INCUBATORS   HEATED   BY  ELECTRICITY  65 

from  joints  and  other  causes,  it  is  apparent  that  the  apparatus  requires  careful 
supervision. 

3.  In  time  the  elasticity  of  the  lamina  diminishes,  with  the  result  that  the 
temperature  is  not  controlled. 

D.  Roux's  incubator. — Roux's  incubator  meets  all  practical  requirements, 
and  has  none  of  the  disadvantages  of  Babes'  and  d'Arsonval's,  and  for  these 
reasons  is  preferable  to  those  instruments. 

The  incubator  consists  of  a  rectangular  wooden  box,  closed  in  front  by  a 
single  or  double  glass  door  raised  on  feet  and  heated  below  by  a  gas  burner. 
The  inside  walls  are  lined  by  a  series  of  vertical  copper  tubes.  The  air  in  the 
incubator  is  maintained  at  a  constant  temperature  by  radiation  from  these 
tubes,  which  are  heated  by  the  gases  from  the  burners  below  passing  up 
through  them.  Ventilation  is  provided  through  holes  in  the  floor  and  roof. 

For  details  of  the  regulator,  see  p.  60. 

Method  of  use. — 1.  Before  using  the  incubator,  it  is  well  to  paste  black  paper  over 
the  doors  to  shield  the  cultures  from  light,  which  may  have  an  adverse  influence  on 
their  growth. 

2.  Lay  a  thermometer  on  each  shelf  to  watch  the  rise  of  temperature.     When 
the  incubator  is  finally  regulated,   each  shelf  has  an  absolutely  constant    tem- 
perature, which  differs  slightly  from  that  of  the  shelf  above  and  below. 

3.  The  tube   C  (fig.  48,  p.   61)  is  connected  to  a  gas  tap,  and  the  tube  D  to  the 
burners  beneath  the  incubator.     Adjust  the  screw  S  controlling  the  piston  E,  and 
turn  it  until  the  latter  is  widely  open. 

4.  Light  the  gas. 

5.  When  the  temperature  on  the  middle  shelf  registers  half-a-degree  below  the 
temperature  required  (36'5°  C.  if  a  temperature  of  37°  C.  is  required),  turn  the 
screw  S  until  the  piston  E  is  closed,  and  the  burner  is  fed  only  by  the  by- pass  V. 
But  when  the  temperature  in  the  incubator  falls,  the  limbs  of  the  metal  U  separate, 
the  rod  T  presses  on  the  piston  rod,  with  the  result  that  the  piston  E  is  opened,  and 
a  larger  volume  of  gas  reaches  the  burner.     The  apparatus  is  now  regulated,  and 
the  temperature  will  remain  constant  without  further  supervision.     If  the  gas  be 
turned  off  at  the  main  and  then  relit,  the  temperature  will  be  regulated  at  the 
height  at  which  it  stood  when  the  gas  was  turned  off.     A  little  vaseline  must  be 
applied  to  the  piston  chamber  from  time  to  time  to  lubricate  the  piston  rod. 

SECTION  III.— INCUBATORS  HEATED  BY  ELECTRICITY. 

When  a  laboratory  has  electric  power  laid  on,  it  may  be  convenient  to  use 
incubators  such  as  those  of  d'Arsonval,  Regaud,  Fouilliaud  [or  Hearson] 
which  can  be  heated  with  electricity. 

A.  D'Arsonval's  electrical  incubator  is  similar  in  appearance  to  Roux's 
incubator.     It  is  fitted  below  with  a  drawer  in  which  the  special  form  of  lamp 
used  for  heating  the  incubator  is  placed.     A  metallic  regulator  is  interposed 
in  the  circuit :    as  the  temperature  rises  it  causes  the  expansion  of  a  metal 
rod,  and  this  breaks  the  circuit  and  cuts  off  the  current.     When  on  the 
other  hand  the  temperature  falls  the  bar  returns  to  its  normal  position  in 
contact  with  a  platinum  point,  and  the  circuit  is  re-established.     To  regulate 

'the  incubator  a  screw  is  turned  until  the  current  passes.  When  a  thermometer 
placed  in  the  incubator  registers  a  few  degrees  below  the  temperature  required, 
the  screw  is  slightly  reversed.  The  temperature  is  noted  again  in  about 
half-an-hour's  time,  and  after  a  few  trials  the  regulation  is  quite  perfect. 

B.  Hearspn's  electrical  incubators.— Hearson's  regulator  (p.  62)  can  be 

applied  to  incubators  heated  by  electricity.  The  circuit  is  broken  and  the 
current  cut  off  when  the  lever  is  raised  by  the  expansion  of  the  fluid  in  the 
capsule. 


66  INCUBATORS 


SECTION  IV.— INCUBATORS   HEATED   BY  PETROL,   GASOLINE, 
OR  PETROLEUM  OIL. 

When  neither  coal  gas  nor  electricity  are  available  as  sources  of  heat,  it 
is  difficult  to  regulate  the  temperature  of  an  incubator  satisfactorily. 

In  these  cases,  incubators  heated  by  petrol  (Lion's,  d'Arsonval-Adnet's, 
Hearson's)  or  by  gas  manufactured  on  the  spot  from  gasoline  (Roux's,  Hear- 
son's),  [or  by  petroleum  oil  (Hearson's)]  should  be  used,  but  they  all  require 
a  good  deal  of  supervision. 


CHAPTER  IV. 

THE   METHODS   OF  SOWING  AND   CULTIVATING 
AEROBIC  ORGANISMS. 

Introduction. 

Section  I. — Instruments  used  for  sowing  cultures,  p.  67. 

Section  II. — The  methods  of  sowing  cultures,  p.  70. 

Section  III. — Conditions  essential  to  satisfactory  growth,  p.  72. 

Section  IV. — The  examination  of  cultures,  p.  73. 

Section  V. — Methods  of  storing  cultures,  p.  75. 

AEROBIC  organisms  should  be  grown  in  vessels  which  while  allowing  free 
access  of  air  at  the  same  time  protect  them  from  dust. 

Various  types  of  culture  vessels  are  in  use  :  for  instance  test-tubes,  circular 
flat-bottomed  flasks,  flasks  of  various  other  shapes,  Petri  dishes,  Soyka 
dishes,  etc. 

Cotton- wool  is  generally  used  as  a  protection  from  dust.  More  rarely 
paper  caps  and  occasionally  glass  covers  are  also  employed. 

In  sowing  a  culture,  the  following  rules  must  be  observed  : 

1.  The  instrument  used  must  be  sterile. 

2.  The  material  to  be  sown  must  be  collected  without  introducing  extraneous 
organisms  : 

3.  And  must  be  transferred  uncontaminated  to  the  medium  it  is  proposed 
to  sow. 


SECTION  I.— INSTRUMENTS  USED   FOR  SOWING  CULTURES. 

Cultures  can  be  sown  with  a  Pasteur  pipette,  platinum  wire,  or  glass 
needle. 

A.  Pasteur  pipettes. 

This  consists  of  a  piece  of  glass  tubing  drawn  out  in  the  blow-pipe,  and 
sealed  at  the  pointed  end,  the  other  end  being  plugged  with  cotton-wool. 
The  pipette  should  be  about  20  to  25  cm.  long. 

A  number  of  these  pipettes  ready  sterilized  should  always  be  at  hand. 

To  make  a  Pasteur  pipette. — 1.  Take  a  piece  of  glass  tubing  of  5  to  7  mm. 
calibre  (the  size  of  a  lead  pencil),  and  with  a  file  mark  it  off  into  lengths 
of  25  cm.  or  thereabouts.  (Glass  tubing  is  sold  in  lengths  of  about  1  metre, 
and  each  length  should  therefore  cut  up  into  four  pieces.) 

2.  Break  the  tubing  by  holding  it  in  both  hands  and  pressing  the  thumbs 
against  the  glass,  one  on  each  side  of  a  file  mark. 


68          CULTIVATION   OF  AEROBIC  MICRO-ORGANISMS 

3.  Round  off  the  cut  ends  in  the  blow-pipe. 

4.  Plug  the  two  ends  of  each  piece  with  cotton-wool,  which  should  pass 
some  distance  into  the  tubing  and  should  also  project  a  few  millimetres  from 
the  open  end  (fig.  54).      This  is  conveniently  done  by  gently  pressing  the 
wool  in  with  some  blunt-pointed  instrument  (the  thin  end  of  a  three-cornered 
file  will  do  very  well). 

5.  Hold  the  middle  of  the  tube  in  a  slightly  inclined  position  and  heat  it 
in  a  medium-sized  blow-pipe  flame,  turning  it  round  and  round  between 
the  thumb  and  index  fingers  until  the  glass  is  soft.     Withdraw  the  tube  from 
the  flame  and  draw  it   out   quickly  into  a  fine  tube  about  30  cm.  long 
(fig.  54,  A).     Divide  it  into  two  in  the  middle  by  melting  it  in  the  tip  of 
the   flame.      This  will  give  two  pipettes  with   the  capillary  end   of   each 
sealed. 

A  certain  amount  of  skill  is  required  to  make  these  pipettes.  Care  should  be 
taken  that  the  tube  is  drawn  out  straight,  and  this  can  best  be  done  by  the  operator 
resting  his  elbows  on  the  table.  The  tube  should  always  be  taken  out  of  the  flame 
before  attempting  to  draw  it,  and  it  should  be  held  horizontally  while  being  drawn. 
The  tube  should  not  be  drawn  too  fine,  otherwise  it  will  be  too  fragile  for  use. 

6.  Place  the  pipettes  with  their  plugged  ends  downwards  in  a  wire  basket 


FIG.  54 — The  method  of  making  pipettes. 

[or  copper  cylinder,  fig.   1],  and  sterilize  them  at  180°  C.   in   the  hot  air 
sterilizer.     They  are  then  ready  for  use. 

Method  of  using  a  Pasteur  pipette. — 1.  Break  off  the  fine  sealed  end  of  the 
pipette  with  a  pair  of  dissecting  forceps  or  between  the  thumb  nail  and  the 
pulp  of  the  index  finger  [it  is  better  to  make  a  light  scratch  with  a  carburundum 
pencil  before  breaking  off  the  point]. 

2.  Pass  the  broken  end  through  the  flame  of  a  Bunsen  burner  or  spirit 
lamp  to  destroy  any  organisms  which  may  happen  to  have  been  deposited 
on  the  surface. 

3.  Dip  the  sterile  end  into  the  fluid  which  is  to  be  used  for  sowing  the 
medium.     The  fluid  will  rise  in  the  tube  by  capillarity,  or  it  can  be  aspirated 
by  slightly  withdrawing  the  wool  in  the  other  end  of  the  tube.     [A  con- 
venient practice  consists  in  slipping  an  india-rubber  teat  over  the  wool- 
plugged  end.     By  pressing  on  the  teat,  air  is  expelled  :  if  the  capillary  end 
be  now  dipped  in  the  fluid  and  the  pressure  on  the  teat  lightly  relaxed,  as 
much  or  as  little  of  the  fluid  as  is  required  can  be  drawn  up  into  the  tube.] 

In  doing  this,  care  must  be  taken  that  the  aspirated  liquid  does  not  soil  the 
wool  plug ;  and  it  is  necessary  also  to  watch  that  bubbles  of  air  are  not  drawn  in. 

4.  Transfer  the  fine  end  of  the  pipette  as  rapidly  as  possible  to  the  medium 
to  be  sown,  and  let  one  or  more  drops  of  the  fluid  fall  on  to  the  medium, 
either  by  its  own  weight  or  by  blowing  gently  through  the  other  end  [or 
by  compressing  the  teat.] 

5.  The  aspirated  fluid  can  be  kept  free  from  contamination  for  an  inde- 
finite time  by  sealing  the  end  of  the  pipette.     Thus,  tilt  the  pipette  gently 
so  that  the  fluid  runs  up  the  tube  ;   heat  the  point  in  a  small  flame  (the  pilot 
flame  of  a  Bunsen),  and  when  the  glass  is  soft,  draw  it  out  with  a  pair  of 
forceps,  and  the  tube  is  completely  closed. 


INSTRUMENTS  FOR  SOWING  CULTURES 


B 


B.  Platinum  wires. 

Platinum  is  to  be  preferred  to  all  other  metallic  wires  because  it  does  not 
oxidize  after  being  heated  to  redness.  The  wire  must  be  suitably  mounted 
in  a  glass  or  metal  handle,  since  on  account  of  its  high  conductivity  it  cannot 
otherwise  be  held  in  the  fingers. 

A  platinum  wire  (German,  ose)  so  mounted  meets  all  requirements.  It  is 
convenient  to  have  three  sizes  of  wire,  stout, 
medium  and  fine  :  each  will  serve  a  special 
purpose  (fig.  55).  The  fine  wire  is  the  most 
generally  useful,  because  it  cools  more  quickly 
than  the  stouter  wires,  and  this  is  an  important 
consideration  in  the  successful  sowing  of  cul- 
tures. At  the  same  time  it  has  very  little 
rigidity  and  is  easily  bent,  so  that  it  cannot  be 
used  for  instance,  to  sow  cultures  which  adhere 
firmly  to  solid  media,  nor  for  sowing  a  rough- 
surfaced  medium  such  as  potato. 

In  the  laboratory  there  should  always  be  at 
hand  : 

A  fine  straight  wire  for  sowing  stab  cultures 
(fig.  55,  A). 

A  stout  wire  whose  point  is  flattened  in  the 
form  of  a  spatula  (fig.  55,  B). 

A  medium-sized  wire,  which  can  be  bent  to 
any  desired  angle  near  its  end  (fig.  55,  C). 

A  fine  wire  bent  into  a  loop  at  the  end  for 
picking  up  a  drop  of  fluid  (fig.  55,  D). 

Method  of  mounting  platinum  wires. — 1.  Take 
a  glass  rod  5-7  mm.  in  diameter,  and  divide  it  into  lengths  of  20-25  cm. 
by  making  a  mark  with  a  file  and  then  breaking  the  rod  at  this  mark  between 
the  thumbs. 

2.  Cut  the  requisite  number  of  lengths  of  platinum  wire  with  a  pair  of 
strong  scissors,  making  each  5-7  cm.  long. 

3.  Take  one  of  the  pieces  of  glass  rod  in  the  left  hand,  soften  one  end  in 
the  blow-pipe,  rotating  it  between  the  fingers  meanwhile.     With  forceps  in 
the  right  hand  pick  up  one  of  the  pieces  of  platinum  wire,  holding  it  about 
15  mm.  from  one  end,  and  heat  this  end  to  a  white  heat  in  the  flame. 

4.  When  the  heated  end  of  the  glass  rod  is  softened,  push  the  hot  end  of 
the  platinum  wire  into  it,  so  that  a  centimetre  or  more  is  embedded  in  the 
rod.     Heat  for  a  few  moments,  [pull  out  slightly]  and  then  allow  to  cool. 

5.  Round  off  the  rough  edge  of  the  other  end  of  the  glass  rod  in  the  flame. 

6.  Then  with  a  pair  of  dissecting  forceps  bend  the  projecting  end  of  the 
wire  into  a  loop  or  at  a  right  angle,  or  flatten  it  with  a  hammer,  as  the  case 
may  be. 

Technique. — 1.  Hold  the  glass  rod  by  its  upper  one-third,  and  pass  the 
other  end  to  which  the  platinum  wire  is  fused  rapidly  through  the  Bunsen 
to  destroy  any  organisms  which  may  be  present  on  the  surface. 

This  sterilization  of  the  glass  rod  should  be  done  rapidly,  because  the  smooth 
surface  of  the  glass  is  quickly  sterilized  and  moreover  does  not  come  into  immediate 
contact  with  the  culture  :  if  the  glass  be  overheated  there  is  a  risk  that  it  may 
crack  at  the  point  where  the  wire  is  fused  into  it. 

2.  Heat  the  wire  to  redness,  and  on  taking  it  out  of  the  flame  let  it  cool 
for  a  few  seconds  in  the  air. 


FIG.  55.— Platinum  wires. 


70          CULTIVATION   OF  AEROBIC  MICRO-ORGANISMS 

The  wire  must  not  be  exposed  to  the  air  any  longer  than  is  necessary  for 
it  to  cool  otherwise  it  may  be  contaminated  by  dust,  and  it  is  because  it  cools 
more  quickly  that  a  fine  wire  is  most  generally  used. 

3.  Pick  up  the  material  to  be  sown  on  the  needle,  and  transfer  it  to  the 
medium  to  be  inoculated. 

4.  When  the  culture  is  sown,  heat  the  platinum  wire  to  redness  to  destroy 
any  organisms  which  may  still  be  present  on  it. 

This  is  particularly  important  when  dealing  with  pathogenic  micro-organisms. 
If  the  needle  be  not  sterilized  immediately,  it  will  soil  the  bench  and  anything  else 
it  may  touch. 

C.  Glass  needles. 

Draw  out  a  piece  of  glass  rod  in  the  same  way  as  the  tubing  was  drawn 
out  when  making  Pasteur  pipettes.  Then  with  a  glass-cutter,  [file,  or  piece 
of  carburundum  pencil]  divide  the  fine  part  squarely  in  the  middle.  In  this 
way  needles  of  any  degree  of  fineness  can  be  made. 

These  needles  are  not  so  easy  to  handle  as  a  platinum  wire,  but  have  the 
advantage  of  being  rigid.  They  are  useful  for  sowing  deep  stab  cultures  in 
gelatin. 

Flame  the  needles  immediately  before  using  them. 


SECTION  II.— THE   METHODS   OF  SOWING   CULTURES. 

A  culture  medium  may  be  sown  from  another  culture,  or  with  water,  dust, 
blood  or  other  material.  The  method  of  collecting  material  differs  of  course 
according  to  the  source  whence  it  is  derived — and  this  will  be  dealt  with 
later  (Chap.  XII.) — but  the  technique  of  sowing  cultures  is  not  affected  by 
these  variations.  Assume  for  the  moment  that  a  sub-culture  is  to  be  sown 
from  an  already  existing  culture,  and  take  as  an  example  a  broth  culture  of 
the  anthrax  bacillus. 

The  process  may  be  divided  into  three  stages. 

(i)     The  opening  of  the  tube  from  which  the  culture  material  is  to  be  taken. 

(ii)    The  removal  of  the  material. 

(iii)  The  sowing  of  the  new  medium.  Here  several  alternatives  present 
themselves.  It  may  be  required  to  sow — 

(a)  Broth,  or  other  liquid  medium. 

(6)  Stroke  cultures  on  agar,  gelatin,  serum  or  potato. 

(c)  Gelatin  stab  cultures. 

(It  is  sometimes  required  to  sow  single  colonies — for  isolating  organisms 
in  pure  culture :  this  will  be  dealt  with  separately  in  a  later  chapter.) 

These  various  problems  will  now  be  considered  seriatim. 

A.  Method  of  sowing  a  liquid  medium. — Broth  may  be  taken  as  a  type  of 
liquid  media. 

1.  Take  a  tube  of  sterile  broth  and  the  tube  containing  the  organism. 
Flame  the  plugs  of  both  tubes  to  burn  off  the  dust  which  has  collected  on 
them.     Loosen  the  plugs  by  screwing  them  round  with  the  thumb  and  index 
finger  of  the  right  hand,  at  the  same  time  slightly  withdrawing  them. 

2.  Place  both  tubes  side  by  side  in  the  left  hand,  holding  them  as  nearly 
horizontally  as  possible.     The  bottom  of  the  tubes  should  rest  in  the  hollow 
of  the  hand,  their  upper  parts  being  grasped  between  the  thumb,  index  and 
middle  fingers. 

3.  Take  a  platinum  loop  between  the  index  and  middle  finger  of  the  right 
hand,  and  sterilize  it  as  already  described. 

4.  While  the  needle  is  cooling,  take  the  plug,  which  has  already  been 


METHODS   OF  SOWING  CULTURES 


71 


loosened,  out  of  the  tube  containing  the  organism  with  the  thumb  and  index 
finger  of  the  right  hand.  Hold  the  plug  between  the  thumb  and  first  finger. 
5.  Introduce  the  platinum  wire  into  the  tube,  being  careful  not  to  let  it 
touch  the  sides  of  the  mouth.  Take  up  a  drop  of  the  culture  fluid  in  the  loop, 
and  withdraw  the  latter  from  the  tube  (fig.  56).  Flame  the  mouth  of  the 

A 


FIG.  56. — Method  of  sowing  a  liquid  medium. 


tube  at  once  to  destroy  any  organisms  which  may  have  settled  on  it  during 
the  process,  and  replace  the  plug  as  quickly  as  possible. 

6.  Take  the  plug  out  of  the  other  tube,  dip  the  loop  into  the  broth,  and 
after  withdrawing  it  flame  the  mouth  of  the  tube  and  replace  the  plug  as 
before. 

7.  Before  laying  the  platinum  loop  on  the  bench,  heat  it  to  redness  to 
destroy  any  organisms  which  may  still  be  adhering  to  it  (in  this  particular 
.case  the  anthrax  bacillus,  an  organism  pathogenic  to  man). 

8.  See  that  the  tubes  have  been  securely  plugged. 

Write  on  the  tube  which  has  been  sown  the  nature  of  the 
organism  and  the  date  when  it  was  sown. 

It  is  often  convenient  to  cover  the  wool  plug  with  a  small  cap.  This 
is  readily  done  by  twisting  over  the  top  a  small  strip  of  paper,  which 
has  been  rolled  round  its  upper  part.  The  details  of  the  culture  can  be 
written  on  this  (fig.  57) :  paper  caps,  however,  are  liable  to  be  inter- 
changed, so  that  it  is  at  least  a  wise  precaution  to  label  the  tube  as 
well.  The  cap  has  the  advantage  that  it  protects  the  wool  from  all 
liability  to  contamination. 

Notes. — It  is  of  the  utmost  importance  that  culture- tubes  which 
have  to  be  opened  should  be  held  in  an  oblique,  nearly  horizontal 
position  so  that  dust  may  not  fall  into  them. 

No  time  should  be  wasted  during  the  sowing  of  cultures,  in  order  to 
minimize  the  chances  of  them  becoming  contaminated. 

Wool  plugs  must  never  be  laid  on  the  bench.  The  part  of  the  plug 
which  goes  into  the  tube  ought  to  be  prevented  from  touching  any- 
thing. 

The  handle  of  the  platinum  needle  should  never  touch  the  medium. 


FIG.  57.— 
Culture-tube 
protected 
with  paper 


B.  Method  of  sowing  stroke  surface  cultures.— As  an  example 
of  this,  the  sowing  of  a  sloped  agar  tube  may  be  described. 

1.  Proceed  as  under  A,  substituting  a  tube  of  sterile  agar  for 
the  sterile  broth. 

2,  3,  4,  5.  As  under  A. 

6.  Remove  the  plug  from  the  agar  tube,  place  the  end  of  the 

wire  on  the  lowest  part  of  the  surface  of  the  agar,  and  draw  it  in  a  straight 
or  zig-zag  line  over  the  medium. 

7,  8.  As  under  A. 

Note. — In  sowing  potato  the  technique  is  the  same  as  above,  but  more  pressure 
must  be  used  in  drawing  the  needle  over  the  surface  and  a  medium  or  stout  wire 
is  desirable. 


72 


CULTIVATION  OF  AEROBIC  MICRO-ORGANISMS 


C.  The  sowing  of  stab  cultures. — The  method  of  sowing  a  stab  culture  in 
gelatin  will  be  described. 

1.  Proceed  as  under  A,  substituting  a  tube  of  sterile  gelatin  for  the  broth  tube. 

2.  Hold  the  two  tubes  in  the  left  hand  thus  :   place  the  culture  from  which 

the  material  is  to  be  taken  in  the  hollow  of 
the  hand,  supporting  it  between  the  thumb  and 
first  finger,  and  keeping  it  as  nearly  horizontal 
as  possible.  Place  the  gelatin  tube  between 
the  first  and  second  fingers,  so  that  it  is  held 
firmly  between  the  dorsal  surface  of  the  index 
finger  and  the  palmar  surface  of  the  second, 
with  the  mouth  pointing  vertically  downwards . 

3.  Hold  the  straight  platinum  wire  in  the 
palm  of  the  right  hand,  leaving  the  thumb 
and  index  finger  free.  Sterilize  the  needle. 

4  and  5.  As  in  A. 

6.  With  the  thumb  and  first  finger  of  the 
right  hand  remove  the  plug  from  the  gelatin 
tube.     Hold  the  already  charged   platinum 
wire  vertically  below  the  mouth  of  the  tube, 
pass  it  into    the   tube   until  it  touches  the 
surface  of  the  gelatin,  let  the  gelatin  tube 
fall  by  its  own  weight  on  to  the  wire  until 
the  latter  touches  the  bottom,  then  withdraw 
it  sharply  (fig.  58). 

7,  8,  9.  Complete  the  operation  as  in  A. 

Notes. — It  is  difficult  by  forcing  the  needle  into  the  gelatin  to  get  a  straight  stab 
which  reaches  to  the  bottom  without  touching  the  sides.  A  satisfactory  stab  will 
be  more  easily  secured  by  allowing  the  gelatin  to  impale  itself  on  the  needle.  The 
tube  must  therefore  be  held  vertically,  and  not  obliquely. 

Gelatin  which  has  been  made  some  time  is  often  cracked :  in  that  case  stand 
the  tube  in  a  water  bath  until  the  medium  is  liquefied,  then  let  it  set,  and  the  gelatin 
will  be  found  to  be  quite  homogeneous  again. 


FIG.  58. — Method  of  sowing  a  stab 
culture. 


SECTION  III.— CONDITIONS  ESSENTIAL  TO  SATISFACTORY  GROWTH. 

To  ensure  growth  taking  place  after  the  medium  has  been  sown,  the  follow- 
ing conditions  must  be  fulfilled  : 

(a)  The  cultures  must  be  freely  exposed  to  the  air  but  at  the  same  time 
be  protected  from  dust.  This  condition  is  readily  satisfied  by  the  use  of  the 
ordinary  wool  plug,  paper  cap,  etc. 

(6)  The  temperature  must  be  kept  constant. 

(c)  As  far  as  possible  light  must  be  excluded. 

The  two  latter  conditions  are  met  by  keeping  the  cultures  in  an  incubator 
(Chap.  III.). 

Some  micro-organisms  grow  only  at  temperatures  above  30°  C., — generally  37° 
or  38°  C., — while  others  only  grow  well  at  temperatures  below  30°  C.  Gelatin 
cultures  of  course  must  not  be  exposed  to  a  temperature  above  20°-22°  C. 

In  the  laboratory  it  is  useful  to  have  three  incubators : 

1.  One  in  which  the  temperature  is  maintained  at  20°-22°  C.  (the  cool  or  gelatin 
incubator). 

2.  A  second  in  which  the  temperature  is  37°-38°  C.  (the  warm  incubator). 

3.  A  third  in  which  the  temperature  can  be  altered  to  meet  special  cases.     Such 
an  incubator  is  required  sometimes  for  cultures  which  need  a  temperature  above 
38°  C.   (39°-41°  C.),  and  at  other  times  for  growing   organisms   at  temperatures 
between  20°  and  37°  C. 


EXAMINATION   OF  CULTURES  73 

(d)  The  medium  must  be  suitable  to  the  needs  of  the  organism  to  be 
grown.  All  organisms  cannot  be  grown  indifferently  on  any  medium ;  for 
while  some  require  a  medium  rich  in  albuminoid  matter,  others  prefer  sugars^ 
glycerin,  etc.,  and  others  again  will  not  grow  on  serum,  or  potato,  and  so  on. 

In  a  later  part  of  the  book,  when  discussing  individual  organisms,  mention 
will  be  made  of  the  media  most  suitable  for  the  growth  of  each  species. 

SECTION  IV.— THE  EXAMINATION  OF  CULTURES. 

Cultures  should  be  examined  daily  or  even  two  or  three  times  a  day,  and 
the  character  of  the  growth,  which  is  of  great  importance  in  determining  the 
species  to  which  an  organism  belongs,  noted. 

Attention  should  be  particularly  directed  to  the  following  points  : 

A.  In  the  case  of  micro-organisms  growing  in  artificial  cultivation,  no 
matter  what  the  medium,  note  : 

1.  (a)  The  optimum  temperature  of  growth,  (b)  the   limits   of  temperature 
within  which  growth  takes  place. 

2.  The  time  when  growth  first  makes  its  appearance. 

B.  When  cultures  are  growing  in  liquid  media,  note  : 

1.  The  mode  of  growth.     Growth  may  produce  : 

(a)  A  distinct  cloudiness  of  the  medium,  which  may  be  either  an  uniform 
cloudiness  or  a  cloudiness  with  a  watered  silk  appearance,  or  sometimes  a 
cloudiness  with  a  surface  pellicle.     In  these  different  cases  flocculent  deposits 
may  ultimately  form,  and  if  so  their  occurrence  should  be  noted. 

(b)  No   distinct  turbidity  of  the  medium.     Under  these  conditions  the 
growth  may  show  :   (a)  a  surface  pellicle,  which  may  be  either  thin  or  thick, 
fatty  or  wrinkled  ;    (/^)  a  ring  of  growth  round  the  wall  of  the  tube  at  the 
surface  of  the  liquid  ;    (7)  flocculent  deposits  in  the  liquid,  which  may  sub- 
sequently precipitate  ;    (8)  fine  granular  deposits,  which  in  some  cases  adhere 
to  the  walls  of  the  tube  and  in  others  fall  to  the  bottom  of  the  medium. 

2.  The  colour  of  the  growth. 

3.  The  production  of  any  smell  during  growth. 

4.  The  development  of  any  new  substances  in  the  medium  (toxins,  indol, 
acid,  ammonia  compounds,  etc.). 

5.  The  presence  or  absence  of  clot  when  grown  in  milk. 

C.  In  the  case  of  stroke  cultures  : 
(i)  On  agar,  potato  or  serum,  note  : 

1.  The  mode  of  growth. 

(a)  The  growth  may  remain  limited  to  the  line  of  sowing,  and  in  this  case 
it  should  be  further  noted  whether  (a)  the  culture  takes  the  form  of  a  delicate 
homogeneous  and  transparent  streak,  or  occurs  as  discrete  colonies ;  or  (/3) 
whether  the  streak  be  thick,  and  if  so  if  it  be  moist,  greasy,  viscous,  dry  or 
wrinkled. 

(b)  The  growth  on  the  other  hand  may  spread  widely  over  the  surface  of 
the  medium,  and  the  nature  of  the  growth,  that  is  to  say  whether  it  be 
moist,  greasy,  viscous,  dry  or  wrinkled,  is  to  be  noted. 

2.  The  colour  of  the  growth.     Whether  the  line  of  growth  or  the  surrounding 
medium  is  pigmented. 

3.  The  production  of  any  odour. 
(ii)  On  gelatin,  note  : 

1,  2,  3.  The/orm,  colour  and  smell  of  the  growth  as  in  the  preceding  cases. 

4.  Whether  the  organism  liquefies  the  medium,  and  if  so,  the  time  at  which 
liquefaction  begins. 


74 


CULTIVATION   OF  AEROBIC  MICRO-ORGANISMS 


D.  In  the  case  of  stab  cultures  in  gelatin,  note  : 

1.  The  mode  of  growth. 

The  following  appearances  are  observed  with  different  species  : 

(a)  A  straight  line  along  the  line  of  sowing  (fig.  59). 


T 


FIG.  59. — Rectilineal  growth.  FIG.  61. — Arborescent  growth. 

FIG.  60.— Tylotate  growth.  FIG.  62.— Surface  growth. 

Stab  cultures  in  gelatin  without  liquefaction. 

(b)  Growth  in  the  form  of  a  nail,  which  may  be  abundant  or  scanty,  and 
more  or  less  marked  at  the  head  of  the  nail  (fig.  60). 

(c)  An  arborescent,  ramifying  growth  (fig.  61). 

(d)  A  growth  strictly  limited  to  the  surface  (fig.  62). 
2.  Whether  liquefaction  occurs  ;   if  so,  note  : 

(a)  The  time  when  it  is  first  observed. 

(b)  The  form  which  the  liquefaction  takes,  whether  it  be  cylindrical,  funnel- 


FIG.  63.— Cup-shaped  liquefaction.         FIG.  65.— Glove  finger  liquefaction. 

FIG.  64. — Funnel-shaped  liquefaction.     FIG.  66. — Cylindrical  liquefaction. 

Gelatin  stab  cultures  with  liquefaction. 

shaped,  in  the  form  of  a  glove  finger  or  of  a  small  cup  (figs.  63  to  66). 
if  an  air  bubble  appears  at  the  top  of  the  growth. 


Note 


METHODS   OF   STORING   CULTURES  75 

3.  The  colour  of  the  growth,  and  whether  the  growth  itself  or  the  medium 
around  it  is  pigmented. 

4.  The  smell  of  the  culture. 


SECTION  V.— THE  METHODS   OF  STORING   CULTURES. 

When  growth  has  ceased,  the  organisms  retain  their  vitality  for  a  certain 
length  of  time  varying  according  to  the  species  from  a  few  days  to  several 
months  and  even  years,  but  ultimately  they  die  and  sub-cultures  sown  from 
them  remain  sterile. 

The  weakening  and  ultimate  disappearance  of  vitality  are  in  a  large  measure 
the  result  of  the  prolonged  action  of  the  oxygen  of  the  atmosphere  on  organisms 
in  an  old  culture  medium,  and  which  are  not  actively  multiplying ;  to  keep 
•organisms  alive  therefore  it  is  necessary  to  sow  them  from  time  to  time  on 
a  new  medium.  But  the  same  result  is  obtained  by  removing  the  organism, 
once  growth  has  finished,  from  the  action  of  the  air ;  this  may  be  done  as 
follows  : 

1.  Sow  a  broth  culture,  incubate  it  at  the  optimum  temperature  of  growth 
until  no  further  development  of  the  organism  takes  place  (the  time  required 
will  obviously  vary  with  different  organisms). 

2.  Take  a  Pasteur  pipette,  and  make  a  constriction  just  below  the  wool 
plug  by  heating  it  in  the  flame  and  drawing  it  out  a  little  (a,  fig.  67). 


FIG.  67. — Method  of  sealing  up  a  culture  in  a  pipette. 

3.  When  the  pipette  has  cooled,  dip  the  narrow  end  with  the  usual  pre- 
cautions  into    the    culture   and   suck   up  the  broth  until    it   reaches   the 
constriction  a. 

4.  Seal  the  pipette  both  at  a  and  at  the  other  end  as  quickly  as  possible 
in  the  blow-pipe. 

In  this  way  a  small  tube  is  obtained,  which  is  filled  with  the  growth  and 
sealed  at  both  ends.  This  should  be  put  away  in  the  dark. 

[In  many  cases  when  the  tubes  or  flasks  containing  the  culture  are  plugged 
with  wool,  it  will  be  quite  sufficient  to  pour  melted  paraffin  wax  over  the  wool 
and  the  lips  of  the  opening  in  order  to  preserve  the  organisms  alive  for  an 
indefinite  period.] 


CHAPTER   V. 

THE   ISOLATION   OF  AEROBIC   MICRO-ORGANISMS 
IN   PURE  CULTURE. 

Introduction. 

Section  I. — Mechanical  methods,  p.  76. 

1.  Dilution,  p.  76.     2.  Dissemination :  (a)  in  liquefied  solid  media,  p.  77  ;  (6)  on  the 
surface  of  a  solid  medium,  p.  81. 
Section  II. — Biological  methods,  p.  83. 

1.  Heat,  p.  84.     2.  Cultivation  at  the  optimum  temperature,  p.  84.     3.  Cultivation 
on  special  media,  p.  85.      4.  Animal  inoculation,  p.  85. 

BEFORE  a  study  of  the  morphology  and  biology  of  any  miero-organism  can 
be  undertaken  the  organism  must  be  obtained  in  pure  culture,  a  culture, 
that  is,  free  from  all  other  organisms  or  as  they  are  technically  called  con- 
taminations. The  first  step,  therefore,  in  a  bacteriological  investigation  will 
be  the  preparation  of  a  pure  culture. 

It  is  obviously  impossible  in  view  of  their  exceedingly  small  size  to  pick  out 
individual  micro-organisms  and  transfer  them  to  tubes  of  culture  media,  so 
that  resort  has  to  be  had  to  more  complicated  methods.  There  are  numerous 
processes  in  everyday  use  for  the  isolation  of  organisms  in  pure  culture  ; 
for  convenience  of  description  these  may  be  divided  into  two  groups  according 
as  to  whether  in  attempting  to  isolate  an  organism  a  purely  mechanical 
method  depending  upon  dilution  and  dissemination  is  relied  upon,  or  whether 
advantage  is  taken  of  the  biological  properties  of  the  organism. 

The  former,  the  mechanical  methods,  will  be  more  useful  when  every  species 
of  organism  present  in  a  given  material  has  to  be  isolated  while  the  latter, 
the  biological  methods,  are  more  especially  applicable  when  a  particular 
organism  of  which  the  chief  characteristics  are  known  beforehand  has  to  be 
isolated  from  material  in  which  it  is  suspected  to  be  present. 

Above  all  in  attempting  to  isolate  micro-organisms  it  is  of  the  first  import- 
ance to  distinguish  between  aerobic  and  anaerobic  species,  for  according  as 
to  whether  the  one  or  the  other  has  to  be  isolated  so  the  cultures  will  have 
to  be  grown  in  the  presence  or  absence  of  air.  In  the  case  of  anaerobic 
organisms  the  methods  of  isolation  will  be  dealt  with  later  (Chap.  VI.).  The 
present  chapter  is  devoted  entirely  to  the  methods  available  for  the  isolation 
of  aerobic  micro-organisms. 

SECTION  I.— MECHANICAL   METHODS. 
1.  Isolation  by  dilution. 

This  method  was  originally  devised  by  Lister  and  extensively  adopted 
by  Nsegeli  and  by  Miquel,  but  is  now  of  very  limited  application.  It  gives- 
very  exact  results  but  occupies  much  time  and  is  exceedingly  tedious. 


MECHANICAL   METHODS 


77 


Suppose  it  be  required  to  isolate  the  organisms  present  in  a  drop  of  water.  Add 
the  water  to  a  tube  A  containing  10  c.c.  of  sterile  broth.  Thoroughly  mix  the  water 
with  the  broth  by  shaking  the  tube.  The  organisms  present  in  the  drop  of  water 
are  now  diluted  in  10  c.c.  of  broth,  and  since  1  c.c.  corresponds  to  20  drops,  each 
drop  of  broth  contains  20  x  10,  i.e.  200  times  fewer  organisms  than  the  drop  of 
water  under  investigation.  Now  transfer  one  drop  of  the  mixture  from  the  tube 
A  to  each  of  a  series  of  tubes  (B,  B',  B",  ...)  containing  broth.  If  the  original 
drop  of  water  contained  200  organisms,  every  drop  of  fluid  in  tube  A  will  contain 
••igfl=l  organism,  so  that  every  drop  transferred  from  A  to  the  series  B,  B',  B", 
etc.,  will  carry  one  organism,  and  that  organism  will  grow  in  the  tube  B,  B',  or  B" 
to  which  it  has  been  transferred  and  will  give  rise  to  a  pure  culture.  But  if  the 
original  drop  of  water  contained  only  50  organisms,  then  only  one  tube  in  four  of 
the  series  B,  B',  B",  etc.  will  give  rise  to  a  pure  culture.  On  the  other  hand, 
suppose  the  drop  of  water  contained  a  larger  number  than  200  organisms,  it  will  then 
be  necessary  to  dilute  further  until  in  fact  one  drop  contains  not  more  than  one 
organism.  Thus  10  drops  from  A  will  be  transferred  to  a  broth  tube  B,  and  a 
series  of  sub-cultures  C,  C',  C",  etc.,  will  be  sown,  each  with  one  drop  of  broth 
from  B. 

2.  Isolation  by  dissemination. 

The  method  of  isolation  by  dissemination  is  due  to  Koch. 

For  its  application  the  use  of  solid  media  is  necessary.  It  may  be  carried 
out  in  one  of  two  ways  :  either  the  medium  may  be  liquefied  and  then  sown, 
or  the  organisms  may  be  distributed  directly  over  the  surface  of  the  medium. 

1.  Dissemination  in  liquefied  solid  media. 

If  it  be  required  to  isolate  all  the  organisms  present  in  a  drop  of  water, 
the  method  would  be  as  follows  :  Transfer  the  drop  of  water  to  a  tube  of 
sterile  gelatin  previously  liquefied  in  the  water  bath,  and  mix  the  water 
and  gelatin  thoroughly  by  rolling 
the  tube  between  the  hands.  The 
organisms  present  in  the  water  will 
now  be  distributed  through  the  gela- 
tin. Pour  the  gelatin  in  a  thin  layer 
on  to  a  sterile  glass  plate,  and  cool 
it  rapidly.  The  organisms  will  be 
scattered  and  held  in  the  layer  of 
gelatin  like  the  almonds  in  a  piece 
of  nougat.  If  the  plate  be  kept  at 
a  suitable  temperature,  each  organ- 
ism will  grow  in  an  isolated  position, 
and  will  give  rise  to  a  colony  com- 
posed of  a  number  of  micro-organisms 
all  derived  from  the  one  organism 
which  originally  settled  in  that 
position,  and  therefore  to  a  pure 
culture  (fig.  68).  It  will  then  be 
easy  to  pick  out  each  colony  separ- 
ately and  sow  it  on  a  new  medium. 

There  are  in  practice  several  ways 
of  carrying  this  out,  but  the  following 
rules  must  always  be  observed : 

1.  After  liquefying  the  gelatin  or 

agar  and  before  sowing  it,   let  it  cool  sufficiently  (to  30°-40°  C.),  to  prevent 
the  organisms  being  killed  by  the  temperature  of  the  medium. 

2.  Avoid  contaminating  the  culture. 

3.  Protect  the  plates  from  dust. 


FIG.  68. — Isolated  colonies  of  micro-organisms 
on  a  gelatin  plate  (two-thirds  natural  size). 


78 


ISOLATION   OF  AEROBIC  MICRO-ORGANISMS 


A.  Petri  dishes.    Method  recommended. 
(i)   Using  gelatin  as  the  culture  medium. 

Apparatus  required. — (a)  Three  Petri  dishes  (fig.  43)  wrapped  in  filter 
paper  [or  packed  in  a  copper  cylinder  (Chap.  I.)]  and  sterilized  in  the  hot 
air  sterilizer  (a  number  of  these  dishes  should  always  be  kept  ready 
sterilized). 

(b)  Three  sterile  Pasteur  pipettes. 

(c)  Three  tubes  of  sterile  gelatin. 

Technique. — 1.  Melt  the  gelatin  by  standing  the  tubes  in  the  water  bath. 

Gelatin  should  never  be  liquefied  by  holding  it  in  a  gas  flame,  because  the  air 
dissolved  in  the  gelatin  will  appear  as  bubbles  in  the  substance  of  the  medium  and 
will  interfere  with  the  subsequent  examination  of  the  plates. 

2.  Take  up  a  drop  of  the  liquid  to  be  examined  in  a  Pasteur  pipette  and 
add  it  to  one  of  the  gelatin  tubes  (dilution  1),  taking  the  necessary  precau- 
tions to  prevent  contamination.     Replace  the  wool  plug  and  mix  thoroughly 
by  rolling  the  tube  between  the  hands. 

Never  shake  tubes  of  media  as  is  done  in  chemical  investigations,  because  it 
gives  rise  to  frothing  and  this  is  highly  inconvenient. 

3.  With  another  pipette  transfer  three  drops  from  the  first  tube  to  another 
tube  of  gelatin  (dilution  2).     Mix  as  before. 

4.  Transfer  three  drops   of  dilution  2  to  the  third    gelatin  tube   (dilu- 
tion 3). 

The  three  tubes  of  gelatin  will  now  contain  each  a  different  number  of  organisms,, 
and,  according  as  to  whether  the  original  material  contained  many  or  few  organisms, 
dilution  3  or  dilution  1  will  give  the  best  results.  Thus  if  the  number  of  organisms 
be  large,  the  colonies  will  be  confluent  in  the  plate  poured  with  tube  1  and  isola- 
tion will  be  impracticable ;  in  that  case  dilutions  2  and  3  will  be  available. 

5.  Take  out  a  Petri  dish  from  its  envelope.     Take  the  plug  out  of  the  first 
gelatin  tube,  and  flame  the  mouth.     Then  lift  the  cover  of  the  Petri  dish^ 
pour  the  gelatin  into  it  and  cover  the  dish  again  as  quickly  as  possible. 

Spread  the  gelatin  in  an  uniform  layer  over  the  surface 
of  the  dish  by  tilting  it  backwards  and  forwards,  put  it  on  a 
cold  and  level  surface  and  allow  it  to  set.  Then  label  it  and 
put  it  in  the  cool  incubator  (20°  C.). 

6.  Pour  plates  with  the  gelatin  in  tubes  2  and  3  in  the 
same  way. 

7.  Examine  the  plates  every  day,  and  without  lifting  the 
cover  note  the  appearance  of  the  colonies  and  their  charac- 
teristics (both  with  the  naked  eye  and  with  the  aid  of  a 
lens).     Remove  a  portion  of  each  colony  for  the  purpose 
of  making  sub-cultures  and  for  microscopical  examination. 

Roux  bottles. — It  is  often  more  convenient  to  use  a  flat  flask,, 
such  as  Roux's  (fig.  69),  instead  of  Petri  dishes.  The  flasks  are 
perhaps  better  than  the  Petri  dishes  because  they  effectually 
prevent  contamination  of  the  medium  and  evaporation  is  reduced 
to  a  minimum. 

Note. — The  gelatin  plate  method  has  some  disadvan- 
tages, and  is  not  available  in  all  cases.  Thus  : 

(a)  Some  organisms  rapidly  liquefy  gelatin,  and  if  such  are  present  in  the 
material  under  investigation  the  experiment  is  liable  to  be  a  failure. 

(b)  It    is    only  applicable   in   cases    of   organisms   growing    at   tempera- 
tures   below  25°  C.     Above  this  temperature  gelatin  ceases    to    be   a   solid 
medium. 


FIG.  69. — Roux 
bottle. 


MECHANICAL  METHODS  79 

(ii)   Using  agar  as  the  culture  medium. 

Consequently,  agar  plates  are  sometimes  used,  especially  when  pathogenic 
micro-organisms  are  being  investigated.  The  technique  is  essentially  the 
same  as  in  the  case  of  gelatin  plates,  but  the  following  points  must  receive 
attention. 

1.  Agar  only  melts  between  90°  and  100°  C.  and  does  not  set  again  until 
it  cools  to  40°  C.     The  agar  tubes  will  therefore  have  to  be  melted  in  boiling 
water  and  then  allowed  to  cool  until  they  can  be  comfortably  held  in  the 
hand. 

2.  The  tubes  must  be  sown  as  above,  but  the  experiment  must  be  done 
quickly  otherwise  the  agar  will  begin  to   solidify  and  the  plates  will  be 
lumpy. 

It  is  a  good  plan  to  have  the  Petri  dishes  standing  on  a  levelling  apparatus  filled 
with  water  at  40°-45°  C.  (see  below)  before  pouring  the  agar,  and  to  cool  the  plates 
slowly  in  order  to  prevent  the  formation  of  lumps  at  the  time  of  cooling  in  the 
dishes. 

3.  Incubate  the  plates  at  37°  C.     The  plates  should  be  packed  into  a 
large  glass  dish  containing  a  few  pieces  of  filter  paper  soared  in  water  [or 
perchloride  solution]  to  prevent  the  medium  drying  up. 

Agar  gelatin  may  be  used  for  cultures  which  are  to  be  incubated  between 
25°  and  35°  C. 

This  agar  plate  method  never  gives  very  good  results,  and  when  agar  has  to  be 
used  for  isolating  organisms  it  is  much  better  to  employ  surface  cultures  (vide 
infra). 

B.  Koch's  plates. 

The  use  of  Koch's  plates  constitutes  an  ingenious  method  of  isolating 
micro-organisms,  but  the  technique  is  complicated  and  difficult  to  carry 
out  under  strictly  aseptic  conditions  for  the  following  reasons : 

1.  The  plates  must  necessarily  be  exposed  to  the  air  for  a  few  seconds  while 
being  manipulated,  and  so  are  liable  to  contamination ;  but  if  they  be  prepared 
quickly  and  in  a  still  atmosphere,  with  no  dust  blowing  about,  this  exposure  is 
not  of  much  moment. 

2.  In  examining  the  plates,  it  is  also  necessary  to  lift  the  cover  of  the  moist 
chamber  and  so  again  expose  the  medium  to  contamination  from   the   air ;    the 
experiment  is  thus  open  to  error. 

The  technique  of  the  method  is  as  follows  : 

Apparatus  required.— 1.  Three  glass  plates  (9  x  12  cm.)  each  wrapped  up  separately 
in  paper  and  sterilized  in  the  hot  air  sterilizer  (a  number  of  these  plates  should 


FIG.  70. — Glass  support  for  plate  cultures. 


always  be  at  hand  ready  for  use).     [As  in  the  case  of  Petri  dishes,  some  bacteri- 
ologists prefer  to  sterilize  the  plates  in  metal  cases.] 

2.  Three  glass  supports  on  which  to  rest  the  plates  (fig.  70). 

3.  Two  large  circular  glass  dishes,  each  about  20  cm.  in  diameter  but  one  rather 
larger  than  the  other  so  that  they  can  be  fitted  together  to  form  a  box. 

4.  A  cooling  table  consisting  of  a  flat  metal  box  the  top  of  which  is  well  polished 


80  ISOLATION   OF  AEROBIC  MICRO-ORGANISMS 

and  covered  by  a  bell  jar  (fig.  71).  The  table  rests  on  screws  which  enable  it  to  be 
levelled  with  the  aid  of  a  small  spirit  level.  Two  lateral  tubes  fitted  to  the  box 
allow  a  stream  of  cold  water  (or  if  agar  is  being  used,  warm  water)  to  be  passed 
through  it,  and  ice  can  also  be  put  into  the  box  if  necessary  through  a  large  opening 
in  the  bottom  closed  with  a  screw  cap. 

5.  Three  tubes  of  melted  gelatin  and  three  Pasteur  pipettes. 

Technique. — 1.  Pour  a  little  perchloride  of  mercury  solution  into  the  large  glass 
•dish,  and  by  rotating  the  dish  wash  every  part  of  its  interior  with  the  antiseptic. 

Lay  two  or  three  thicknesses  of  filter  paper  in  the  bottom  of  the  dish  and  saturate 


FIG.  71. — Cooling  stand  for  plate  cultures. 


them  with  perchloride  solution.     (This  constitutes  a  moist  chamber,  the  object  being 
to  prevent  the  gelatin  plates  drying  up.) 

Wash  the  glass  stands  (fig.  70)  with  perchloride,  and  place  one  of  them  in  the 
bottom  of  the  dish. 

2.  Place  the  cooling  stand  on  the  operator's  left,  level  it  and  fill  it  with  cold   or 
iced  water.     Wipe  the  top  carefully  to  remove  any  dust  that  may  be  on  it,    and 
wash  the  inside  of  the  glass  cover  with  perchloride  solution. 

3.  Sowthe  three  tubes  of  gelatin,  1,2,  and  3,  as  in  the  gelatin  plate  method  (p.  78  A  (i)). 

4.  Take  one  of  the  glass  plates,  tear  off  the  paper  cover  along  one  of  the  edges, 
hold  it  by  one  of  its  corners  between  the  thumb  and  first  finger  of  the  right  hand 
[or  better  in  a  pair  of  sterile  forceps],  slightly  raise  the  glass  cover  with  the   left 

hand,  and   lay  the  plate  on  the  glass    support 
already  placed  there.     Replace  the  glass  cover. 

5.  Take  the  plug  out  of  tube  3,  flame  the  upper 
2  or  3  cm.  of  the  tube,  and  then  while  not  raising 
the  glass  cover  more  than  is  necessary  introduce 
the  mouth  of  the  tube  beneath  it,  pour  the  gelatin 
on  to  the  centre  of  the  glass  plate  and  spread  it 
with  the  upper  flamed  part  of  the  tube.     With- 
draw the  tube,  replace  the  bell   jar,  and  allow 
the  gelatin  to  set. 

6.  When  the  gelatin  has  set,  raise  the  bell  jar 
again,  take  hold  of  the  glass  plate  by  one  of  its 
corners,  transfer  it  as  quickly  as  possible  to  the 
moist  chamber  (the  cover  of  which  is  raised  with 

the  left  hand,  after  replacing  the  bell  jar),  and  lay  it  on  the  glass  stand. 

Bridge  the  glass  plate  with  the  second  glass  stand,  and  replace  the  cover  of  the 
moist  chamber. 

It  will  be  noticed  that  the  gelatin  has  not  come  in  contact  either  with  the  walls 


FIG.  72. — Arrangement  of  the  glass 
plates  in  a  moist  chamber. 


MECHANICAL  METHODS  81 

of  the  moist  chamber  or  with  the  glass  stands,  and  this  explains  why  perchloride 
can  be  used  for  sterilizing  these  pieces  of  the  apparatus. 

7.  In  the  same  way  pour  a  plate  with  tube  No.  2,  place  it  in  the  moist  chamber, 
and  put  the  third  glass  support  in  position. 

8.  Repeat  the  process  with  tube  No.  1,  and  place  it  on  the  third  plate-rest. 

9.  Put  the  moist  chamber  in  the  incubator  at  20°  C.     The  plates  have  been  arranged 
so  that  the  one  containing  the  largest  number  of  organisms  is  nearest  the  top  of  the 
chamber.     Colonies  will  appear  on  it  earlier  than  on  the  other  plates,  and  it  can  be 
examined  and  studied  without  touching  the  latter,  which  should  not  be  interfered 
with  until  growth  appears  on  them. 

C.  Esmarch's  tubes. 

Apparatus  required. — 1.  Three  Pasteur  pipettes. 

2.  Three  tubes  of  gelatin.     The  tubes  should  be  rather  longer  and  wider 
than  ordinary  culture-tubes,  and  each  should  contain  10  c.c.  of  sterile  gelatin. 

3.  Three  sterile  india-rubber  caps. 

Technique. — 1.  Sow  the  tubes  1,  2,  and  3  as  before  (p.  78  A  (i)). 

2.  Slip  an  india-rubber  cap  over  the  wool  plug  of  each. 

3.  Cool  each  tube  in  turn  under  the  cold  water  tap  :    hold  it  as  nearly 
horizontally  as  possible,  so  that  the  gelatin  coats  the  whole  of  the  inner 
surface  of 'the  tube  below  the  plug  (but  without  touching  the  wool),  and 
rotate  it  between  the  index  finger  and  thumb  of  each  hand.     When  the 
gelatin  sets  it  is  thus  spread  in  a  thin  layer  over  the  whole  of  the  inner  surface 
of  the. tube  and  forms  a  "  roll  "  tube. 

4.  When  the  gelatin  has  set,  take  off  the  india-rubber  cap  and  incubate 
the  tubes  at  20°  C. 

This  method  has  the  great  advantage  of  absolutely  preventing  any  contamination 
of  the  medium,  but  the  investigation  of  the  colonies  which  develop  is. rendered 
more  difficult  by  the  cylindrical  shape  of  the  gelatin  surface. 

2.   Dissemination  on  the  surface  of  a  solid  medium. 

When  it  is  necessary  to  isolate  the  organisms  present  in  a  non-liquid  pro- 
duct such  as  a  false  membrane,  viscous  sputum,  etc.,  a  small  portion  of  the 
material  is  smeared  over  the  surface  of  some  solid  medium  contained  in  a 
Petri  dish  or  sloped  in  a  tube.  This,  which  is  the  method  now  universally 
adopted  for  the  isolation  of  diphtheria  bacilli  from  false  membranes,  is 
available  when  the  media  which  it  is  proposed  to  use  cannot  be  liquefied 
by  heat,  e.g.  potato  or  serum. 

If  there  be  reason  to  suppose  that  the  material 
under  investigation  is  very  rich  in  organisms 
(excreta,  for  example),  a  small  portion  is  diluted 
in  a  few  cubic  centimetres  of  broth  or  sterile 
water  and  a  trace  of  the  dilution  used  for 
sowing  cultures.- 

Two  methods  are  available. 

A.  Stroke  cultures. 

The  method  of  isolation  on  agar  plates  will 
be  taken  as  an  illustration  (fig.  73). 
Apparatus  required. — 1.  A  medium  or  stout 

i    ,  FIG.  73.— Isolation  of  organisms 

platinum  Wire.  by  parallel  stroke  culture  on  Petri 

2.  A  tube  of  agar.  disheg.    xi 

3.  A  sterile  Petri  dish. 

Technique. — 1.  Melt  the  agar  and  pour  it  with  the  usual  precautions  into 
the  Petri  dish. 


82  ISOLATION   OF  AEROBIC  MICRO-ORGANISMS 

Let  the  agar  set  firmly. 

2.  Take  up  a  trace  of  the  material  under  investigation  on  the  wire,  raise 
the  cover  of  the  Petri  dish,  and  without  recharging  the  needle  make  a  series 
of  parallel  strokes  on  the  agar  each  a  few  millimetres  distant  from  the  other. 

As  the  needle  is  drawn  over  the  agar  the  material  on  it  is  transferred  to 
the  latter,  and  it  is  obvious  that  after  the  wire  has  been  drawn  across  the 
agar  three  or  four  times  the  number  of  organisms  left  along  the  line  of  any 
stroke  will  be  but  few  in  number. 

3.  Incubate  the  plate  at  37°  C.     The  colonies  which  develop  along  the 
first  strokes  will  be  very  numerous,  but  will  be  fewer  and  fewer  along  the 
later  ones. 

B.  Surface  cultures. 

1,  Classical   method. — This   method   of   isolation   may  be  illustrated  by 
describing  it  as  it  would  be  used  with  sloped  serum,  but  the  method  is  the 
same  for  agar,  potato,  etc. 

Apparatus  required. — 1.  A  stout  platinum  wire  flattened  at  the  end. 

2.  Three  tubes  of  sloped  solidified  serum. 

Technique. — 1.  Take  up  a  trace  of  the  material  under  investigation  on 
the  wire. 

2.  Remove  the  plug  from  one  of  the  serum  tubes,  dip  the  needle  into  the 
tube  and  smear  the  whole  surface  of  the  medium,  commencing  below  and 
working  towards  the  mouth  (tube  1). 

3.  Without  recharging  the  needle  sow  a  second  tube  of  serum  in  the  same 
way  (tube  2). 

4.  Sow   the   third   tube   similarly,  again   without   recharging   the   needle 
(tube  3). 

5.  Incubate  the  tubes  at  37°  C. 

As  the  result  of  drawing  it  over  the  surface  of  the  serum,  the  needle  is 
gradually  wiped  clean  of  the  organisms  with  which  it  was  charged  and 
which  have  been  deposited  on  the  serum.  Tube  No.  1  will  grow  numerous 

confluent  colonies,  but  tubes  No.  2  and  No.  3 
will  grow  fewer  colonies  and  some  of  them  will 
be  well  isolated.  The  discrete  and  isolated 
colonies  on  the  latter  tubes  can  be  used  for 
further  investigation. 

2.  Veillon's  method. — 1.  Take  a  trace  of  the 
material  on  a  platinum  wire. 

2.  Without  recharging  it,  dip  it  into  the  water 
of  condensation  -at  the  bottom  of  4  or  6  agar 
tubes. 

3.  Replug  the  tubes  and  sow  the  surfaces  of 
the  agar  by  running  the  water  of  condensation 
over  them.     Incubate  the  tubes  in  the  vertical 
position. 

3.  Chantemesse's  method. — This  is  useful  for 
?IG>  7bylrSn'rm0efthodaniS  the  purpose  of  isolating  organisms   present  in 

stools. 

1.  Dilute  a  trace  of  the  material  in  several  cubic  centimetres  of  distilled 
water. 

2.  Dip  a  sterile  badger-hair  pencil  into  this  highly  diluted  material. 

3.  Brush  the  surface  of  a  series  of  5  or  6  agar  plates  (prepared  in  Petri 
dishes  as  described  above  under  A,  p.  8D  without  recharging  the  brush. 


BIOLOGICAL   METHODS  83 

4.  Incubate  the  plates  at  37°  C. 

Chantemesse  adopts  this  method  in  isolating  the  typhoid  bacillus,  using  a  special 
medium  instead  of  agar  (Chap.  XXI.). 

[4.  Burri's  method. — The  aim  of  the  method  is  to  grow  a  colony  from  a 
single  organism.  A  dilute  emulsion  of  the  organism  is  made  and  further 
diluted  in  an  emulsion  of  indian  ink ;  small  drops  of  the  latter  are  then  laid 
on  the  surface  of  gelatin,  covered  with  a  cover-glass  and  examined  under  the 
microscope.  Those  cover-glasses  which  cover  only  a  single  organism  are  then 
transferred  to  other  media  and  incubated. 

[Apparatus  required. — 1.  A  sterile  emulsion  in  water  of  commercial  indian 
ink,1  or  better  a  1  in  9  emulsion  in  water  of  a  colloidal  compound  known  as 
Pelikan  Tusche  No.  54.2 

2.  A  number  of  sterile  Petri  dishes,  slides  and  cover-glasses. 

3.  Half  a  dozen  ready  poured  gelatin  plates  in  large  Petri  dishes. 

4.  Sterile  dissecting  forceps. 

5.  Two  or  three  fine  drawing  pens  also  sterilized. 

[Technique. — 1.  Prepare  a  dilute  and  homogenous  emulsion  in  normal 
saline  solution  of  the  culture  or  material  under  examination. 

2.  Place  a  sterile  slide  in  one  of  the  Petri  dishes ;  with  a  small  platinum 
loop  put  four  drops  of  the  indian  ink  emulsion  in  a  row  on  the  slide  and 
replace  the  cover  of  the  dish. 

3.  With  a  straight  platinum  wire  transfer  a  small  drop  of  the  bacterial 
emulsion  to  the  left-hand  drop  (No.  1)  of  the  indian  ink  on  the  slide  and  mix 
intimately.     Transfer  similarly  a  small  drop  from  No.  1  to  No.  2  and  mix ; 
from  No.  2  to  No.  3  and  so  on. 

4.  With  one  of  the  sterile  drawing  pens  take  up  the  right-hand  drop  of 
bacterial-indian  ink  emulsion  and  lay  it  in  a  series  of  very  minute  droplets 
on  the  surface  of  one  of  the  gelatin  plates.     Cover  each  drop  separately  with 
a  sterile  cover-glass. 

5.  Disseminate  similarly  drop  No.  3  on  another  plate  and  cover  as  before. 

6.  Examine  the  droplets  under  the  microscope  using  a  dry  lens  and  an 
high  eyepiece.      If  necessary  an  oil  immersion  lens  may  be  used  ;    in  that 
case   place  a  drop   of  oil  on  the  upper  surface  of  the   cover-glass.     The 
organisms  will  be  seen  as  bright  objects  on  a  dark  background. 

7.  When  a  droplet  is  found  in  which  only  a  single  organism  is  suspended, 
raise  the  cover-glass  with  a  pair   of   sterile  forceps — the    indian   ink   and 
organism  will  be   found  to  adhere  to  the  cover-glass — and  transfer  it  to 
another  plate  of  gelatin  or  some  other  suitable  medium  laying  the  cover- 
glass  drop  side  downwards.     Incubate.] 

SECTION   n.— BIOLOGICAL   METHODS. 

The  methods  now  to  be  described  are  only  available  when  the  detection 
and  isolation  of  a  given  organism  is  in  question,  and  depend  upon  a  know- 
ledge of  one  or  more  properties  of  the  organism  ;  this  knowledge  is  applied 
to  facilitate  the  growth  of  that  organism  while  at  the  same  time  hindering 
the  growth  of  any  others  which  may  be  present. 

The  separation  of  anaerobic  from  aerobic  organisms  may  be  quoted  as 
an  example  of  the  principles  involved.  Aerobic  organisms  cannot  grow  in 
the  absence  of  free  oxygen  ;  so  that  by  sowing  the  material  in  an  atmosphere 
free  from  oxygen  cultures  of  anaerobic  organisms  alone  are  obtained. 

The  methods  most  generally  in  use  will  be  described. 

1  Giinther,  Vienna.  2  Grtibler,  Leipzig. 


84  ISOLATION   OF  AEROBIC  MICRO-ORGANISMS 

1.  The  application  of  heat  to  the  isolation  of  micro-organisms. 

Spore-forming  organisms  can  resist  temperatures  of  80°  to  100°  C.  and 
even  105°  C.  for  several  minutes,  but  non-spore-bearing  organisms  are  soon 
destroyed  when  heated  to  about  60°  C.  Hence  it  will  be  easy  to  separate  a 
spore-bearing  from  a  non-spore-bearing  organism  in  a  mixture  containing 
both ;  it  will  only  be  necessary  to  heat  the  mixture  for  a  few  minutes  to  a 
temperature  between  80°  and  105°  C.,  according  to  the  resistance  of  the 
spore,  and  subsequently  to  sow  it  in  a  tube  of  broth.  Thus  a  pure  culture 
of  the  anthrax  bacillus  can  be  obtained  by  heating  an  impure  culture  to 
'80°-85°  C.  for  5  minutes. 

An  infusion  of  hay  if  heated  to  100°  C.  for  10  minutes  will  give  a  pure 
culture  of  the  Bacillus  sublilis.  Similarly  an  infusion  of  potato  chips  incubated 
for  two  or  three  days  and  then  heated  to  105°  C.  for  5  minutes  will  give 
a  pure  culture  of  the  potato  bacillus,  and  so  on. 

In  carrying  out  the  above  experiments,  it  is  necessary  to  work  with  fluid  cultures 
or  suspensions,  since  organisms  when  dried  or  mixed  with  solid  matter  are  much 
more  resistant  to  heat.  It  is  further  essential  to  the  success  of  the  method  that  all 
parts  of  the  culture  fluid  be  raised  to  the  required  temperature,  otherwise  some 
of  the  non-sporing  forms  will  escape  destruction  and  the  experiment  will  be  only 
a  partial  success. 

The  technique  is  as  follows  : 

1.  Prepare  a  very  fine  Pasteur  pipette  with  a  constriction  below    the 
wool  (p.  75). 

2.  Fill  the  pipette  with  the  culture  up  to  the  constriction  and  seal  both 
ends  in  the  flame. 

3.  Immerse  the  tube  in  a  water  bath  heated  to  the  temperature  required, 
and  leave  it  for  5  or  10  minutes.     If  the  temperature  required  be  above 
100°  C.  the  tube  must  be  heated  in  the  autoclave. 

4.  Dry  the  tube  and  then  break  off  one  end  with  a  pair  of  sterile  forceps 
after  passing  it  through  the  flame.     Withdraw  a  little  of  the  fluid  into  another 
sterile  pipette,  being  careful  to  avoid  contaminating  it,  and  sow  sub-cultures. 

2.  Isolation  by  cultivation  at  the  optimum  temperature. 

Fractional  cultivation. 

While  some  organisms  will  grow  at  any  temperature  between  10°  and 
40°  C.,  the  limits  of  temperature  within  which  growth  takes  place  are  in 
the  majority  of  cases  much  more  restricted.  Thus  a  large  number  of 
saprophytes  grow  slowly  and  poorly  above  30°  C.  ;  many  of  the  pathogenic 
bacteria  attain  their  maximum  development  between  30°  and  40°  C.,  others 
will  not  grow  below  30°  C.,  while  yet  another  group  (the  typhoid-colon  group) 
grows  at  43°  C. — a  temperature  which  is  too  high  for  the  multiplication  of 
most  micro-organisms.  These  facts  with  regard  to  differences  in  the  optimum 
temperature  at  which  micro-organisms  grow  are  applied  for  the  purpose  of 
isolating  organisms  in  pure  culture. 

For  example,  the  colon  bacillus  can  be  isolated  from  stools  by  sowing  a 
trace  of  the  material  in  broth  and  incubating  at  43°  C.  Incubation  at  this 
temperature  however  does  not  at  once  yield  a  pure  culture,  for  the  organisms 
which  were  present  with  the  colon  bacillus  in  the  original  material  have  not 
been  destroyed  but  their  growth  merely  arrested  ;  so  that  were  a  sub-culture 
to  be  sown  from  this  first  broth  culture  and  incubated  at  37°  C.  these  co- 
existing organisms  would  multiply  under  the  more  favourable  conditions  and 
contaminate  the  culture  of  the  colon  bacillus.  To  eliminate  them  the  method 
of  fractional  cultivation  may  conveniently  be  adopted ;  thus  when  the  first 


BIOLOGICAL   METHODS  85 

broth  culture  incubated  at  43°  C.  has  become  cloudy  a  trace  of  it  is  sown  in 
another  tube  of  broth  which  is  then  similarly  incubated  at  43°  C.,  and  from 
the  second  tube  a  third  is  sown,  and  so  on,  until  after  several  sub-cultures 
in  series,  each  incubated  at  43°  C.,  a  pure  culture  of  the  colon  bacillus  is 
ultimately  obtained. 

A  method  analogous  to  this  is  employed  when  it  is  required  to  isolate  the  cholera 
vibrio  from  specifically  infected  stools,  only  in  this  particular  case  the  action  of 
temperature  (37°-38°  C.)  is  combined  with  that  of  a  special  medium  (vide  infra)  in 
which  fractional  cultivation  is  effected.  This  will  be  found  to  be  in  most  cases  quite 
a  useful  method  for  eliminating  saprophytic  organisms. 

3.  Isolation  by  cultivation  on  special  media. 

The  growth  of  any  given  organism  to  the  exclusion  of  that  of  others  which 
are  present  with  it  may  be  effected  by  sowing  the  material  on  a  medium 
which  is  designed  to  meet  the  requirements  of  the  organism  to  be  isolated. 

The  diphtheria  bacillus,  for  instance,  can  be  isolated  in  pure  culture  by 
smearing  the  surface  of  a  number  of  serum  tubes  with  a  piece  of  membrane. 
Isolation  in  this  case  is  favoured  by  the  fact  that  serum  is  very  well  adapted 
to  the  growth  of  the  diphtheria  bacillus,  but  more  or  less  unfavourable  to  the 
multiplication  of  organisms  which  are  generally  found  associated  with  this 
bacillus. 

For  the  isolation  of  the  cholera  vibrio,  Koch  and  Metchnikoff  recommend 
special  media  which  though  of  poor  nutritive  value  happen  to  meet  its 
particular  requirements.  Thus  a  trace  of  the  "  rice  water  "  stool  is  sown 
in  a  tube  of  Metchnikoff's  liquid  peptone-gelatin  medium  (p.  33)  and  incu- 
bated at  38°  C.  Under  these  circumstances  the  growth  of  the  cholera  vibrio 
is  much  more  rapid  than  that  of  the  other  organisms  present.  The  vibrio 
being  a  very  strictly  aerobic  organism  forms  a  pellicle  on  the  surface  of  the 
liquid,  and  if  after  the  culture  has  been  incubating  for  12  hours  or  so, 
a  trace  of  the  film  be  examined,  it  will  be  found  to  consist  of  an  almost  pure 
culture  of  the  cholera  vibrio.  To  further  purify  the  culture  recourse  must 
be  had  to  fractional  cultivation  [sowing  the  sub-culture  with  a  trace  of  the 
pellicle  taken  up  on  the  point  of  a  fine  wire],  and  three  passages  will  be  all 
that  is  necessary  before  finally  plating  out  on  gelatin  as  described  on  p.  78. 

[For  the  isolation  of  bacilli  of  the  typhoid-colon  group  MacConkey  has 
introduced  bile-salt  media.  The  material  suspected  to  contain  the  organism 
is  sown  in  a  liquid  bile-salt  medium,  and  after  incubation,  preferably  at 
42°  C.,  a  trace  of  the  culture  is  plated  out  on  a  bile-salt-agar  and  suspicious 
colonies  picked  off  for  further  examination  (for  fuller  details  of  the  method, 
see  Chaps.  XXI.  and  XXIII.).] 

Finally,  in  some  cases,  the  growth  of  associated  organisms  may  be  arrested 
by  adding  to  the  medium  some  antiseptic  which  is  not  injurious  to  the 
organism  to  be  isolated.  Chantemesse  for  instance  advises  the  use  of  media 
containing  carbolic  when  attempting  the  isolation  of  the  colon  or  typhoid 
bacillus,  and  Eisner  suggests  the  use  of  potassium-iodide-gelatin  for  the 
same  purpose. 

As  has  been  indicated  above,  this  and  the  method  of  cultivation  at  the 
optimum  temperature  may  be  combined;  Vincent  for  instance  adopted  a 
combination  of  the  two  methods  in  his  attempts  to  isolate  the  typhoid 
bacillus  (Chap.  XXI.). 

4.  Isolation  by  animal  inoculation. 

In  some  cases  the  simplest,  and  perhaps  the  only,  method  of  isolating  a 
pathogenic  organism  in  pure  culture  from  material  in  which  it  is  mixed  with 


86  ISOLATION   OF  AEROBIC  MICRO-ORGANISMS 

non-pathogenic  species  will  be  to  inoculate  the  material  into  a  suitable 
animal. 

For  example,  to  isolate  the  pneumococcus  from  pneumonic  sputum  a 
little  of  the  latter  may  be  inoculated  beneath  the  skin  of  a  mouse  ;  the  animal 
will  soon  die  and  its  blood  will  be  found  to  contain  a  pure  culture  of  the 
pneumococcus. 

Similarly,  to  isolate  the  bacillus  of  malignant  oedema  from  soil  in  which 
there  is  also  present  a  large  number  of  other  organisms,  a  little  of  the  earth 
is  rubbed  up  into  a  thin  emulsion  in  a  few  drops  of  sterile  water  and  inocu- 
lated beneath  the  skin  of  the  abdomen  of  a  guinea-pig.  The  animal  dies 
from  an  infection  known  as  Pasteur's  septicaemia,  and  the  serous  peritoneal 
exudate  will  contain  the  bacillus  in  pure  culture. 

Many  opportunities  of  studying  this  method  of  isolating  organisms  will 
occur  later. 


CHAPTER  VI. 

THE  CULTIVATION   AND   ISOLATION  OF  ANAEROBIC 
MICRO-ORGANISMS. 

Introduction. 

Section  I. — The  methods  of  abstracting  air  from  culture  media,  p.  87, 

1.  By  boiling,  p.  87.     2.  By  displacing  the  air  with  some  inert  gas,  p.  88.     3.  By 
absorbing  the  oxygen,  p.  89.     4.  By  the  use  of  a  vacuum,  p.  90. 
Section  II. — The  cultivation  of  anaerobic  organisms,  p.  92. 

1.  Liquid  media,  p.  92.     2.  Solid  media,  p.  99. 
Section  III. — The  isolation  of  anaerobic  organisms,  p.  101. 

1.  Plate  method,  p.  101.     2.  Tube  method,  p.  103. 
Section  IV. — Vacuum  incubators,  p.  104. 

SOME  organisms  grow  equally  well  under  both  aerobic  and  anaerobic  con- 
ditions, others  grow  only  when  the  medium  in  which  they  are  sown  contains 
no  trace  of  free  oxygen  ;  the  former  are  known  as  the  facultative  anaerobes, 
the  latter  as  the  strict  anaerobes.  The  cultivation  of  the  strictly  anaerobic 
organisms  is  accompanied  by  certain  technical  difficulties  arising  out  of  the 
necessity  for  removing  all  traces  of  air  from  the  culture  medium  in  which  they 
are  sown.  The  culture  media  are  the  same  for  the  two  classes,  but  for  the 
strictly  anaerobic  organisms  special  forms  of  culture  apparatus  and  special 
methods  are  required,  and  it  is  to  a  description  of  these  that  the  present 
chapter  is  devoted. 

The  recent  investigations  of  Tarrozzi,  which  have  been  confirmed  by 
Wrzosek,  Guillemot,  Ori  and  others,  seem  to  show  that  oxygen  does  not 
directly  exert  any  harmful  influence  on  anaerobic  organisms,  but  that  the 
presence  of  free  oxygen  prevents  the  media  furnishing  the  nutritive 
substances  necessary  for  anaerobic  life. 

Anaerobic  organisms  can  in  fact,  as  Tarrozzi  has  shown,  be  grown  in 
presence  of  the  oxygen  of  the  atmosphere  by  simply  adding  pieces  of  animal 
tissue  or  some  reducing  agent  to  the  culture  media  (vide  infra). 

SECTION  I.     METHODS   OF   ABSTRACTING  AIR  FROM   CULTURE 

MEDIA. 


1.  By  boiling. 

can  be  expellee 
must  be  boiled  f 
and  then  be  cooled  rapidly  away  from  the  air. 


Gases  dissolved  in  a  liquid  can  be  expelled  by  boiling.     To  expel  all  the 
air  from  a  culture  medium  it  must  be  boiled  for  20  minutes  to  half  an  hour, 


88       CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 


2.  By  displacing  the  oxygen  of  the  atmosphere  by  an 

inert  gas. 

The  air  in  a  liquid  can  be  displaced  by  passing  a  current  of  an  inert  gas 
through  it.  Hydrogen,  carbonic  oxide,  nitrogen  and  ordinary  coal  gas 
have  all  been  suggested  for  the  present  purpose. 

A.  Hydrogen. — For  the  growth  of  anaerobes,  hydrogen  is  preferable  to  the 
other  gases  mentioned.  Not  only  is  it  easily  prepared,  but  it  has  no  injurious 
effect  on  the  organisms. 

A  convenient  form  of  apparatus  for  readily  obtaining  a  continuous  supply 
of  hydrogen  is  that  illustrated  in  fig.  75.  The  bottle  A  contains  a  1  in  6 


FIG.  75. — Apparatus  for  yielding  a  continuous  supply  of  hydrogen. 

solution  of  pure  sulphuric  acid  in  water.  The  bottle  B  contains  some  pieces 
of  broken  glass  at  the  bottom,  and  above  this  a  layer  of  granulated  zinc. 
By  simply  raising  the  bottle  A  and  opening  the  tap  R  a  stream  of  hydrogen 
will  issue  from  the  tube  T  ;  similarly,  by  closing  the  tap  R  and  lowering  A 
to  the  level  of  B  the  supply  is  stopped.  To  remove  impurities,  especially 
oxygen,  it  is  desirable  to  wash  the  hydrogen  as  it  issues  from  the  bottle  B 
by  passing  it  through  the  following  solution  : 

Caustic  potash,  50  per  cent,  in  water,         -  -         50  c.c. 

Pyrogallol,      -  1  gram 

contained  in  a  wash-bottle  F. 

It  is  even  better  to  have  two  wash-bottles,  one  containing  a  solution  of  potassium 
permanganate  slightly  acidified  with  sulphuric  acid,  the  other  a  solution  of  potas- 
sium permanganate  made  slightly  alkaline  with  caustic  soda.  These  solutions 
must  be  frequently  renewed.  The  method  is  particularly  useful  for  removing 
traces  of  hydrocarbons  and  phosphides  and  arsenides  of  hydrogen. 

The  hydrogen  before  being  passed  through  the  culture  medium  should 
be  tested  by  means  of  indigo  white,  to  ascertain  that  it  is  quite  free  from 
traces  of  oxygen  (p.  92). 

[Hydrogen  is,  however,  most  conveniently  obtained  by  keeping  a  cylinder 
of  the  compressed  gas  in  the  laboratory.  Cylinders  of  the  gas  can  be  obtained 


ABSTRACTION   OF   AIR   FROM   CULTURE   MEDIA          89 

in  commerce  guaranteed  to  contain  99'6  per  cent,  of  hydrogen,  the  remaining 
O4  per  cent,  being  almost  if  not  entirely  composed  of  air,  which  represents 
O08  per  cent,  of  oxygen.  When  used  for  the  cultivation  of  anaerobic 
organisms  in  a  Bulloch's  apparatus  (pp.  96  and  100) — which  is  the  method 
usually  adopted  in  England — the  gas  requires  no  preliminary  washing, 
but  is  passed  direct  from  the  cylinder  into  the  bell  jar  containing  the 
cultures.] 

B.  Carbonic  anhydride. — Carbonic  anhydride  is  harmful  to  a  large  number 
of  organisms,  and  its  use  for  that  reason  is  not  to  be  recommended  in  the 
present  connexion.      The  apparatus  described  above  for  the  preparation  of 
hydrogen  can  be  utilized  for  the  preparation  of  the  gas,  if  pieces  of  white 
marble  be  substituted  for  the  zinc,  and  hydrochloric  acid  for  the  sulphuric 
acid.     The  gas  should  be  washed  by  passing  it  through  a  solution  of  sodium 
hydrosulphite  contained  in  the  wash-bottle  F  (fig.  75). 

C.  Nitrogen. — The  preparation  of  this  gas  is  so  difficult  that  its  use  should 
be  abandoned  in  practical   bacteriology.     [Nitrogen  can  however  now  be 
obtained  as  a  commercial  product  in  the  form  of  cylinders  of  the  compressed 
gas,  which  on  analysis  is  found  to  contain  very  little  oxygen.     In  our  experi- 
ence the  results  obtained  with  this  compressed  gas  in  the  growth  of  anaerobic 
organisms  have  been  quite  satisfactory.] 

D.  Coal  gas. — The  use  of  coal  gas  is  not  to  be  recommended  in  anaerobic 
methods,   because  many  of  the  component  gases  comprising  the  mixture 
are  inimical  to  micro-organisms. 

Note. — Before  passing  any  gas  into  a  culture  medium  it  must  be  sterilized  by 
filtration  through  a  sterile  cotton- wool  plug.  The  technique  of  this  operation  will 
be  referred  to  later. 

3.  By  absorbing  the  oxygen. 

A.  Advantage  may  be  taken  of  the  affinity  possessed  by  some  substances 
for  combining  with  oxygen  to  remove  the  latter  from  culture  media.     In 
practice  oxygen  is  generally  absorbed  by  resting  the  culture-tube  on  a  glass, 
or  metal,  support  inside  a  much  larger  tube  (about  20  to  25  cm.  in  length), 
and  then  pouring  the  following  solution  into  the  latter  : 

Pyrogallol,      -  1  gram. 

Alcoholic  potash,     -  1       „ 

Water,  -  -         10  c.c. 

Plug  the  outer  tube  with  a  tightly-fitting  india-rubber  bung.  Under 
these  conditions  oxygen  diffuses  through  the  wool  plug  of  the  inner  culture- 
tube  and,  being  absorbed  by  the  pyrogallol,  turns  the  solution  brown. 

Sellards,  using  a  similar  apparatus,  substitutes  fragments  of  phosphorus  for  the 
potassium  pyrogallate  solution. 

B.  In  some  cases  it  will  be  found  convenient  to  add  to  the  medium  some 
easily  oxidizable  substance,  which  does  not  interfere  with  the  growth  of  the 
organism ;  e.g.  glucose  (2  per  cent.),  formate  of  soda  (0'5  per  cent.),  sodium 
sulphindigotate  (0*1   per  cent.),   fragments  of  tissue,   etc.     This  method  is 
generally  adopted  in  the  case  of  deep  stab  cultures  in  agar  (vide  infra). 

C.  By  sowing  the  surface  of  an  anaerobic  culture  in  a  solid  medium  with 
some  aerobic  organism  which  absorbs  a  good  deal  of  oxygen,  air  can  be 
prevented  from  reaching  the  anaerobic  culture,  the  growth  of  the  latter 
taking   place  beneath  the  growth  of  the  aerobic  organism  (Roux).     This 
method  will  be  described  in  detail  when  dealing  with  stab  cultures. 


90       CULTIVATION   OF   ANAEROBIC   MICRO-ORGANISMS 


4.  By  the  use  of  a  vacuum. 

The  use  of  apparatus  by  means  of  which  a  vacuum  can  be  produced 
simplifies  the  methods  of  cultivating  anaerobes  and  at  the  same  time 
renders  them  more  exact ;  and  moreover,  as  both  a  mercury  pump  and 
water  pump  are  in  everyday  use  in  the  laboratory,  the  essentials  are 
ready  to  hand.  The  use  of  a  vacuum  is  generally  supplemented  by 
washing  with  an  inert  gas ;  by  the  combination  of  the  two  methods 
it  should  be  possible  to  remove  all  trace  of  oxygen  from  the  culture 
vessels. 

In  many  laboratories  the  further  precaution  is  taken  of  adding  some  oxygen- 
absorbing  solution,  generally  pyrogallol  and  potash,  to  absorb  any  traces  of  oxygen 
which  might  still  remain. 

The  reason  for  washing  with  an  inert  gas  lies  in  the  physical  fact  that  two  gases, 
which  do  not  enter  into  chemical  combination,  rapidly  diffuse  when  brought  in 
contact  and  form  an  uniform  and  constant  mixture.  The  rate  of  diffusion  varies 
directly  as  the  differences  in  density  of  the  gases  ;  the  greater  the  difference  the 
more  rapid  the  diffusion. 

In  practice  it  is  impossible  to  obtain  a  perfect  vacuum,  so  that  after  exhausting 
a  vessel  full  of  air  a  residuum  of  air  remains.  Now  if  the  vessel  be  filled  with 
hydrogen  and  exhausted  again  the  residuum  will  consist  of  a  mixture  of  air  and 
hydrogen  ;  by  repeating  the  process  several  times,  the  amount  of  air  ultimately 
present  will  be  infinitesimal  in  amount. 

Suppose  that  after  exhausting  a  vessel  of  2  litres'  capacity  there  remains  1  c.c. 
of  air  measured  at  atmospheric  temperature  and  pressure ;  fill  the  vessel  with 
hydrogen,  and  the  1  c.c.  of  air  will  be  diluted  1  in  2000  ;  exhaust  again  until  only 
1  c.c.  remains,  and  the  residual  gas  will  contain  o0Jo(T  c.c.  of  air  and  £$$$  c.c.  of 
hydrogen  ;  after  a  second  washing  with  hydrogen  the  volume  of  air  will  not  exceed 

4.000,000  c'c> 

A.  Mercury  pump. — With  this  apparatus  an  almost  perfect  vacuum  can 
be  obtained,  but  it  is  expensive  and  being  delicate  is  liable  to   be  easily 
damaged  ;  moreover  time  and  skill  are  required  to  use  it  to  the  best  advantage. 
Its  use  is  limited  in  practice  to  very  delicate  investigations  and  to  vessels  of 
small  capacity.     Without  going  into  the  details  of  the  working  of  the  pump 
the   following   points   of    importance   in   connexion  with   its    use    may   be 
noted. 

1.  The  pump  must  always  be  tested  to  see  that  it  is  working  properly 
and  that  the  taps  fit  well.     Any  taps   not  fitting  tightly  must  be  lubri- 
cated. 

2.  Connect  the  vessel  containing  the  culture  to  the  pump,  and  exhaust 
until  there  is  a  wide  difference  between  the  levels  of  the  mercury  in  the  two 
limbs  of  the  manometer. 

3.  Then  open  the  tap  connected  to  the  hydrogen  supply  just  a  little,  and 
let  the  hydrogen  pass  slowly  into  the  receiver  until  the  mercury  has  reached 
its  original  position. 

4.  Turn  off  the  supply  of   hydrogen.     Exhaust   again,    and   repeat   the 
process  two  or  three  times. 

5.  Seal  the  neck  of  the  culture  vessel  in  the  flame  in  vacuo. 

B.  Water  pump. — On  account  of  its  moderate  price  and  of  the  ease  with 
which  it  is  worked,  a  water  pump  is  much  more  often  used  for  producing 
a  vacuum  than  a  mercury  pump.     The  vacuum  is  only  approximate,  and 
exhaustion  with  a  water  pump  must  therefore  be  combined  with  washing 
with  an  inert  gas. 

The  pump,  which  is  best  made  of  metal  (d'Alvergniat's  pattern),  should 


ABSTRACTION   OF   AIR   FROM   CULTURE   MEDIA 


91 


consist  of  a  copper  pipe  fitted  with  a  manometer  M  and  joining  the  pump 
proper  at  a  right  angle  T,  as  shown  in  the  figure  (fig.  76). 


FIG.  76.— A  water  pump  with  its  fittings. 

A   water-pressure   of   about  two  atmospheres  is  necessary.     The  method 
of  exhausting  and  washing  is  as  follows  : 

1.  By  means  of  pieces  of   thick-walled  rubber  tubing  (pressure-tubing), 
connect  the  vessel  containing  the  culture  to  the  tap  R",  and  the  hydrogen- 
generating  apparatus  to  the  tap  R7.  ^ 

Close  the  taps  R  and  R',  leaving  R"  open  throughout 
the  experiment. 

2.  Turn  on  the  water  tap  E,  gradually  open  R,  and  watch 
the  manometer  needle. 

3.  When  the  vessel  has  been  exhausted  as  completely  as 
possible,  close  R,  and  by  gradually  opening  R',  fill  the 
culture  vessel  with  hydrogen. 

4.  When  the  manometer  needle  has  fallen  to  zero,  close 
R',  open  R  again,  and  exhaust  the  vessel  a  second  time. 

5.  After  exhausting  and  washing  with  hydrogen  two  or 
three  times,  seal  the  neck  of  the  culture  vessel  in  the  flame 
in  vacuo. 

It  is  not  always  necessary  to  wash  with  hydrogen,  but  if 
exhaustion  alone  be  relied  upon  the  culture  liquid  should  be 
boiled  ;  this  can  easily  be  done  by  very  slowly  raising  the  tem- 
perature to  30°-35°  C.  either  by  holding  the  vessel  in  the  hand 
or  by  standing  it  in  a  vessel  of  luke-warm  water  or  by  heating 
it  with  a  small  flame. 

Note. — In  using  a  water  pump  the  tap  R  must  be  closed, 
so  as  to  cut  off  all  connexion  between  the  water  and  the 

culture,  before  turning  off  the  water  tap 
at  the  end  of  an  experiment.  If  this  pre- 
caution be  omitted,  the  vacuum  will  induce 
a  violent  rush  of  water  into  the  culture 
vessel. 

A  similar  inrush  of  water  will  also  occur 
if  from  any  cause  whatever  the  pressure 
in  the  main  is  suddenly  lowered  during 
the  process  of  exhausting ;  consequently 
a  bottle  of  2  or  3  litres'  capacity,  and  fitted 
up  as  shown  in  fig.  77,  should  always  be 
interposed  between  the  pump  and  the 
vessel  to  be  exhausted. 

If  then  there  be  a  rush  of  water,  it  will 
collect  in  the  bottle  and  will  not  contami- 
nate the  culture.    It  is  even  better  to  use  a 
FIG.  78.-water  pump  with  safety  reservoir,    pump  fitted  with  a  brass  reservoir  (fig.  78), 


FIG.  77. —  Wash- 
bottle  fitted  up  for  use 
with  a  water  pump  to 
prevent  backflow  of 

10  the  culture 


92       CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 

which  will  act  in  the  same  way  as  the  bottle  and  prevent  a  rush  of  water 
into  the  culture.  The  only  ground  of  objection  to  this  piece  of  apparatus  is 
that  of  cost. 

Tests  for  oxygen. 

It  is  often  necessary  to  know  whether  a  gas — the  hydrogen  used  in  washing 
anaerobic  cultures,  for  instance — is  free  from  oxygen.  This  may  be  deter- 
mined by  passing  the  gas  through  a  solution  of  indigo  white,  a  substance 
which  turns  blue  in  presence  of  small  quantities  of  oxygen. 

Indigo  white  is  prepared  by  treating  indigotine  (pure  indigo)  with  concentrated 
sulphuric  acid.  This  solution  when  neutralized  with  sodium  carbonate  gives 
sodium  sulphindigotate,  which  in  presence  of  an  excess  of  alkali  is  easily  decolourized 
by  reducing  agents.  Sodium  sulphindigotate  is  generally  reduced  with  sodium 
hydrosulphite,  obtained  by  adding  to  powdered  zinc  a  concentrated  solution  of 
sodium  bisulphite  saturated  with  sulphurous  anhydride.  Sodium  hydrosulphite 
is  a  powerful  reducing  agent  and  decolourizes  the  indigo,  combining  with  the 
oxygen  of  the  atmosphere  to  form  bisulphite. 

The  gas  may  therefore  be  tested  for  oxygen  by  bubbling  it,  away  from  air, 
through  a  solution  of  indigo  white. 

To  make  sure  that  a  culture  medium  contains  no  free  oxygen,  a  few  drops 
of  a  O2  per  cent,  solution  of  sulphindigotate  of  sodium  may  be  added  until 
the  colour  is  distinctly  blue,  then  1  per  cent,  by  weight  of  a  normal  soda 
solution  and  1  per  cent,  of  glucose.  When  all  the  free  oxygen  has  been 
removed  the  blue  colour  disappears,  the  glucose  reducing  the  indigo  under 
these  conditions. 

If  a  culture  medium  tinted  with  a  few  drops  of  a  solution  of  sodium 
sulphindigotate  be  sown  with  an  anaerobic  organism  and  freed  from  oxygen, 
the  blue  colour  will  be  destroyed  as  growth  of  the  organism  proceeds, 
decolourization  commencing  in  the  immediate  neighbourhood  of  the  colonies. 
The  micro-organism  takes  the  oxygen  necessary  for  its  growth  from  the 
substances  around  it,  and  acts  therefore  as  a  reducing  agent. 

SECTION  II.— THE  CULTIVATION  OF  ANAEROBIC  ORGANISMS. 
1.  Liquid  media.  * 

A.  Pasteur's  method. — This  is  the  method  originally  employed  in  growing 
anaerobic  organisms.  It  is  now  only  of  historical  interest. 

A  large  round  flask  A  (fig.  79)  with  two  tubulures 
is  filled  with  broth  :  the  tubulure  B  dips  into  a  porce- 
lain dish  three-parts  filled  with  the  same  liquid.  The 
tap  R  being  closed,  the  flask  and  porcelain  dish  are 
simultaneously  heated  to  boiling  for  half  an  hour. 
The  dissolved  air  is  thus  driven  off.  The  apparatus 
is  allowed  to  cool  in  situ,  and  then  the  end  of  the 
tube  B  is  transferred  to  a  vessel  full  of  mercury.  The 
funnel  E  is  filled  with  carbonic  acid  gas,  and  then 
(away  from  air)  with  .the  fluid  to  be  sown.  The  tap 
R  is  next  opened  and  the  fluid  runs  into  the  flask, 

_      care  being  taken  that  a  little  remains  in  the  funnel 

FIG.  79.— Pasteur's  original  method  to  prevent  access  of  air  to  the  flask.  The  culture  is 
for  the  cultivation  of  anaerobic  or-  then  incubated. 


B.  Roux's  pipette.    Method  recommended.™ 

1.  Make  a  constriction  in  a  sterile  Pasteur  pipette  in  a  small  flame  of  the 
blow-pipe  just  below  the  cotton-wool  plug  (fig.  67  a,  p.  75). 

2.  After  flaming  the  point  of  the  pipette,  break  it  off,  dip  it  into  a  tube  of 
broth  already  sown  with  the  organism  to  be  cultivated  and  aspirate  the 
broth  into  the  pipette  until  the  latter  is  three-parts  full. 


CULTIVATION  IN   LIQUID   MEDIA 


93 


3.  Tilt  the  pipette  so  as  to  raise  the  point  and  seal  the  latter  in  a  small 
flame. 

4.  Connect  the  other  end  to  an  exhaust  pump.     Exhaust  and  wash  with 
hydrogen  alternately. 

It  is  often  sufficient  when  a  vacuum  is  established  to  boil  the  liquid  as  described  at 
p.  91.  When  the  pipette  is  heated  even  very  slightly  the  liquid  will  boil  violently  and 
will  tend  to  pass  into  the  aspirating  tube  ;  this  may  be  prevented  by  first  heating  the 
upper  part  of  the  tube  above  the  liquid. 

5.  Seal  the  pipette  at  the  constriction  a,  in  vacuo.     Dip  the  ends  of  the 
pipette  into  Golaz's  wax  to  strengthen  them.     Incubate. 

6.  When  the  culture  has  grown,  flame  and  break  the  end  a  of  the  pipette 
and  withdraw  the  culture  by  means  of  a  Pasteur  pipette. 

C.  Pasteur,  Joubert  and  Chamberland's  tube.— With  this  apparatus  two 
successive  cultures  can  be  sown  without  exposing  the  medium  to  the  air 
while  sowing  the  second  culture. 

It  consists  (fig.  80)  of  an  inverted  U-tube,  each  limb  of  which  is  provided  with  a 
lateral  tubulure  terminating  in  a  fine  point.  A  third  tubulure  originates  from  the 
convexity  of  the  U,  and  this  is  constricted  in  two  places  a  short  distance  apart, 
and  plugged  with  wool  between  the  two  constrictions. 


FIG.  80. — Pasteur,  Jou- 
bert and  Chamberland's  tube 
for  the  cultivation  of  anae- 
robic organisms. 


FIG.  81. — Pasteur's  tube 
for  the  cultivation  of  anae- 
robic organisms. 


FIG.  82.-.-Lacpmme'stube 
for  the  cultivation  of  anae- 
robic organisms. 


1.  Plug  the  vertical  part  C  with  wool  between  the  constrictions  c  and  c]?  seal  the  points 
of  the  lateral  tubulures  a  and  b  and  sterilize  the  tube  in  the  hot  air  sterilizer. 

2.  When  it  has  cooled,  flame  the  lateral  tube  a,  break  off  its  point  and  dip  the  latter 
into  the  broth,  which  has  been  sown  beforehand.     Aspirate  the  liquid  into  the  limb  A 
by  applying  suction  to  C.     Seal  up  the  end  of  a  in  the  flame  again. 

3.  Flame  the  lateral  tube  6,  break  off  its  point,  dip  the  end  into  a  tube  of  sterile  broth, 
and  aspirate  the  broth  into  the  limb  B.     Seal  the  end  of  b  in  the  flame. 

Note. — In  carrying  out  the  second  and  third  operations,  be  careful  that  the  liquids 
in  the  two  limbs  do  not  mix.  The  limbs  should  not  be  more  than  one-third  filled. 

4.  Attach  the  upper  end  of  C  to  the  exhaust  pump.     Exhaust  the  air,  and  wash  two 
or  three  times  with  hydrogen.     Seal  the  tube  at  the  constriction  c  in  vacuo. 

5.  Incubate  the  tube  in  the  vertical  position.     Growth  will  occur  in  A,  while  the  broth 
in  B  will  remain  clear  and  serve  as  a  control. 

6.  When  growth  in  A  has  ceased,  tilt  the  apparatus  so  that  a  drop  or  two  of  the  culture 
passes  from  A  into  the  sterile  broth  contained  in  B.     Incubate  again,  and  growth  will 
now  take  place  in  B. 

D.  Pasteur's  tube. — This  is  a  more  simple  form  of  the  preceding,  and 
consists  of  a  single  limb  of  the  U-tube  just  described  (fig.  81). 

After  sterilizing  the  tube  aspirate  the  broth,  already  sown  with  the  organism,  through 
the  narrow  tube  a,  seal  the  point  of  a  in  the  flame,  exhaust  through  B,  seal  this  tube 
at  b  and  incubate. 

Lacomme's  tube  (fig.  82)  is  a  modification  of  Pasteur's;  it  is  used  in  an  exactly  similar 
manner,  and  is  cheaper. 


94        CULTIVATION   OF   ANAEROBIC   MICRO-ORGANISMS 

E.  Long-necked   flask    method. — This    is    a    useful    method    when    large 
quantities  of  culture  are  required. 

1.  Take  a  flask  with  a  long  neck,  fill  it  one-third  full  of  broth,  plug  with  wool 
and  autoclave  (fig.  83). 

2.  When  cool  take  out  the  wool  plug  and  sow  the  broth  with  a  long  Pasteur 
pipette,    taking    every    care    to    avoid   introducing   contaminations.     Replace   the 
plug  and  push  it  half-way  down  the  neck. 

3.  Make  a  shallow  constriction  below  the  plug  at  A,  and  draw  out  the  upper 
end  B. 

4.  Connect  the  upper  end  B  with  the  water  pump :    exhaust,  and   wash   with 
hydrogen :  seal  the  neck  above  the  plug  in  the  flame  in  vacuo,  and  incubate. 

5.  To  withdraw  the  culture  after  incubation,  cut  the  neck  above  the  level  of 
the    plug    (p.   47),   take  the  plug   out,   and  aspirate  the  fluid  into  a  pipette  or 
into  a  sterile  distributing  flask.     [The  culture   may    equally    well   be    drawn  up 
into  a  Cobbett's  bulb  by  the  method  used  in  the  preparation  of  serum  (p.  45).j 

F.  Bottle  method.    Method  recommended. — The  advantages  of  the  method 
are  that  (1)  large  quantities  of  broth  can  be  used,  and  (2)  the  culture  can  be 
very  easily  removed. 

1.  Select  a  bottle  of  1  or  2  litres'  capacity  with  a  mouth  large  enough  to 
take  an  india-rubber  bung  perforated  with  two  medium-sized  holes.  Fill 
the  bottle  two-thirds  full  of  broth  (fig.  84). 


FIG.  83. — Flasks  with  long  necks 
for  the  cultivation  of  anaerobic 
organisms. 


FIG.  84. — Bottle  arranged  for  anaerobic 
cultivation. 


2.  Take  a  piece  of  glass  tubing  of  the  same  diameter  as  the  holes  in  the 
india-rubber  bung,  and  bend  it  at  right  angles  about  its  centre.     In  one 
limb  make  two  constrictions  a  short  distance  apart,  and  plug  this  space  with 
wool.     Pass  the  other  limb  through  one  of  the  holes  in  the  bung  so  that  its 
lower  end  projects  a  distance  of  3  or  4  cm.  below  the  bung. 

Take  another  piece  of  glass  tubing  and  bend  it  as  shown  in  the  figure. 
Pass  the  straight  limb  through  the  other  hole  so  that  it  almost  reaches  the 
bottom  of  the  bottle,  while  the  other  limb  terminates  outside  the  bottle  in  a 
solid  point  sealed  in  a  flame. 

3.  Fit  the  india-rubber  bung  firmly  into  the  neck  of  the  bottle  and  sterilize 
at  115°  C.  for  20  minutes,  but  let  the  temperature  rise  gradually  for  fear  of 
cracking  the  bottle. 


CULTIVATION   IN   LIQUID   MEDIA 


95 


4.  When  the  apparatus  has  cooled,  ascertain  that  the  bung  fits  firmly,  and 
then  lute  the  joints  between  the  bung  and  the  neck  of  the  bottle  and  between 
the  tubes  and  the  bung  with  Golaz's  [or  paraffin]  wax.     Dry  the  wool  plug 
in  A  by  gently  heating  the  glass  tube  in  a  Bunsen  burner. 

5.  In  order  to  sow  the  broth,  flame  the  external  limb  of  B,  and  break  off 
the  point  with  sterile  forceps.     Dip  the  end  into  the  tube  containing  the 
organism  to  be  cultivated;  aspirate  a  few  drops  into  the  bottle  through  A. 
and  seal  the  point  of  B  in  the  flame. 

6.  Connect  A  to  the   water  pump.     Exhaust   and  wash  with  hydrogen 
several  times,  keeping  the  lower  two-thirds  of  the  bottle  in  a  bath  of  water 
at  35°-40°  C. 

7.  Seal  A  in  the  flame  in  vacuo  at  the  constriction  beyond  the  wool  plug. 
Incubate. 

After  incubating  for  2  or  3  days  the  gas  produced  as  the  result  of  the  growth 
of  the  organism  accumulates  to  such  an  extent  as  to  prevent  further  multiplica- 
tion. At  this  stage  it  is  well  to  break  off  the  sealed  end  of  A  (leaving  the  cotton- 
wool plug  in  position,  of  course)  to  allow  the  pent-up  gases  to  escape  ;  the  pressure 
of  the  gases  remaining  in  the  bottle  and  continuously  generated  by  the  growth  of 
the  organism  is  sufficient  to  prevent  the  entrance  of  air. 

It  is  as  well  to  add  a  little  calcium  carbonate  or  tricalcium  phosphate  to  the 
medium  before  sterilizing  it,  because  with  some  organisms  the  amount  of  acid  produced 
is  so  considerable  as  very  soon  to  interfere  with  and  perhaps  altogether  check  the 
growth.  If  these  salts  be  added,  the  acids  will  be  neutralized  as  they  are  formed. 

8.  To  withdraw  the  culture  from  the  bottle,  flame  the  end  of  B  and  break 
off  the  point.     Blow  through  A  and  collect  the  culture  in  a  sterile  flask. 

G.  Pyrogallol  method.  Buchner's  tube.— 1.  Boil  a  tube  of  sterile  broth, 
cool  rapidly,  and  sow. 

2.  Place  this  tube  as  already  described  at  p.  89  inside  a  larger  tube  containing 
a  solution  of  potassium  pyrogallate,  and  incubate  (fig.  85). 


FIG.  85. — Buchner's 
tube  for  growing  anae- 
robic organisms. 


FIG.  86. — Turro's  tube. 


Turro's  tube.— This  method  has  advantages  over  Buchner's  in  that  the  oxygen  is 
much  more  rapidly  absorbed  and  the  culture  is  visible  during  incubation  (fig.  86). 

1.  Pour  the  medium  (broth,  agar  or  gelatin)  into  A  through  the  narrow  tube  a. 
Plug  the  upper  end  of  the  apparatus  with  an  india-rubber  stopper  C  and  autoclave. 


96        CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 


2.  When  it  has  cooled,  sow  the  medium  through  a  ;    then  with  a  pipette  fill  the 
bulb  B  one- third  full  of  potassium  pyrogallate. 

3.  Replace  the  stopper  C  and  lute  it  with  paraffin  or  Golaz's  wax.     Tilt  and 
rotate  the  apparatus  so  that  the  pyrogallate  runs  all  over  the  surface  of  the  bulb 
to  accelerate  the  absorption  of  oxygen.     Incubate. 

[H.    Bulloch's   method.    Method   recommended. — Bulloch's  method  is   a 
modification  of  the  preceding,  designed  to  allow  of  the  incubation  of  a  number 


of  plates  or  tubes  at  one  time.  The  principle  is  the  same  and  depends  upon 
the  absorption  of  oxygen  by  pyrogallate  of  potassium.  The  apparatus  con- 
sists of  a  circular  glass  bell  jar  (fig.  87)  flanged  below,  with  two  openings 
above,  each  of  which  is  fitted  with  a  ground-glass  stopper  prolonged  into  a 


Y/////A 


FIG.  88. — A  modified  form  of  Bulloch's  apparatus. 


glass  tube  bent  at  right  angles  and  fitted  with  a  closely  fitting  tap.  One  of 
these  tubes  only  passes  a  few  centimetres  into  the  bell  jar  while  the  other 
reaches  nearly  to  the  bottom. 

[Fig.   88    shows    a    slightly  modified  and  less  fragile  form  of  Bulloch's 


CULTIVATION   ON  LIQUID  MEDIA  97 

apparatus.  The  openings  are  laterally  situated  and  are  plugged  with 
india-rubber  corks,  each  of  which  is  perforated  by  a  piece  of  glass  tubing. 
To  each  of  the  latter  a  piece  of  red  rubber  pressure-tubing  is  attached  at  its 
outer  end.  When  the  apparatus  is  to  be  closed,  the  tubing  is  compressed  by 
screw  clips  and  a  piece  of  glass  rod  tightly  fitted  into  its  distal  end. 

[1.  On  the  ground-glass  plate  stand  a  shallow  glass  vessel  3-4  inches  deep,  but 
having  as  large  a  diameter  as  will  permit  of  the  bell  jar  being  placed  over  it. 

[2.  Place  about  J  oz.  of  pyrogallol  in  the  bottom  of  the  vessel. 

[3.  Place  the  tubes  in  a  suitable  receptacle,  and  stand  the  latter  on  a  glass 
tripod  in  the  vessel  containing  the  pyrogallol. 

[4.  Grease  the  lower  flanged  end  of  the  bell  jar  with  unguentum  resinae, 
and  press  it  firmly  down  on  to  the  ground-glass  plate  in  such  a  way  that 
the  long  tube  passes  into  the  shallow  glass  vessel. 

[5.  Aspirate  about  40  c.c.  of  strong  potash  solution  (30-40  per  cent.)  into 
the  vessel.  Then  screw  up  the  clips  as  tightly  as  possible,  and  plug  the  distal 
end  of  the  tubing  with  glass  rod. 

[6.  Incubate. 

[7.  To  remove  the  tubes,  withdraw  the  pieces  of  glass  rod,  gently  unscrew 
the  clips,  slide  the  bell  jar  off  the  glass  plate  and  lift  out  the  receptacle 
containing  the  tubes.] 

[I.  Bullock's  method  modified.— Method  recommended. — The  use  of  pyro- 
gallol and  potash  is  as  a  rule  supplemented  by  exhaustion  and  washing 
with  hydrogen. 

[1.  Proceed  as  in  1,  2,  3  and  4  above. 

[2.  Attach  the  glass  tube  which  passes  just  inside  the  apparatus  to  a 
water  pump  connected  with  a  manometer,  and  the  other  tube  which  dips 
into  the  vessel  to  a  cylinder  of  hydrogen. 

[3.  Exhaust  the  vessel. 

[4.  Turn  on  the  hydrogen  tap  and  pass  a  slow  stream  of  gas  until  the 
manometer  falls  to  zero.  Unless  this  be  carefully  done  the  pressure  of 
hydrogen  will  lift  the  bell  jar. 

[5.  Turn  off  the  hydrogen.     Exhaust  again. 

[6.  Wash  with  hydrogen  again,  and  again  exhaust. 

[7.  Screw  up  both  clips  as  tightly  as  possible. 

[8.  Disconnect  the  bell  jar  from  the  water  pump  and  also  from  the  cylinder 
of  hydrogen. 

[9.  Connect  the  tubing  that  dips  into  the  vessel  to  a  beaker  containing  a 
40  per  cent,  solution  of  potash  in  water.  Gently  loosen  the  clip  and  allow 
40  c.c.  or  so  of  the  solution  to  enter  the  bell  jar,  being  careful  to  allow  no 
air  to  enter.  Screw  up  the  clip. 

[10.  Insert  a  tightly-fitting  piece  of  glass  rod  into  each  piece  of  india-rubber 
tubing  on  the  distal  side  of  the  clip. 

[11.  The  vessel  is  now  ready  to  be  placed  in  the  incubator.] 

[The  security  of  the  joints  should  be  tested  on  the  following  day,  or  even  later 
on  the  same  day.  To  do  this,  attach  the  same  piece  of  tubing  as  before  to  the 
manometer,  turn  on  the  water  pump  to  exhaust  the  rubber  connexions,  etc.,  and 
then  loosen  the  screw  clip.  If  the  apparatus  is  securely  fastened  the  mercury 
should  remain  at  the  same  level  as  when  the  bell  jar  was  exhausted.] 

J.  Legros'  method.  Method  recommended. — By  this  method  the  air  is 
excluded  from  the  medium  by  means  of  a  layer  of  vaseline  oil.  Pour 
sufficient  oil  into  the  culture-tube  to  form  a  layer  5-10  mm.  deep  on  the  sur- 
face of  the  medium.  Plug  with  wool,  and  autoclave.  Sow  in  the  ordinary 
way  through  the  layer  of  oil. 


98       CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 

In  the  case  of  media  which  cannot  be  heated  strongly,  Ch.  Nicolle  recommends 
the  following  modification  of  the  method:  Pour  sufficient  sterile  vaseline  oil  into 
the  flask  or  tube  containing  the  medium  to  form  a  thin  layer  on  the  surface.  Stand 
the  culture  vessel  in  a  water  bath  at  40°  C.  and  connect  the  mouth  of  the  vessel  to 
a  water  pump.  After  exhausting  the  whole  of  the  dissolved  air  the  culture  medium 
is  protected  from  air  by  the  layer  of  oil. 

K.  Rosenthals  method.  Method  recommended. — 1.  Distribute  the  medium 
(broth,  milk,  etc.)  into  tubes.  Pour  lanolin  previously  liquefied  by  heat  into 
each  tube  so  that  it  forms  a  layer  15  mm.  thick  on  the  surface.  Plug  the 
tubes  with  wool  and  autoclave  at  120°  C.  After  sterilization,  cool  the  tubes 
rapidly  in  a  vertical  position. 

Tubes  prepared  by  this  method  can  be  kept  for  about  two  months.  If  kept 
longer  than  this,  it  is  well  to  heat  them  to  100°  C.  for  15  minutes  before  sowing 
them.  It  is  an  advantage  to  use  tubes  slightly  constricted  about  the  middle ;  the 
medium  occupies  the  lower  part  of  the  tube  up  to  the  constriction,  while  the 
lanolin  fills  the  constricted  part.  Any  gas  which  may  be  formed  easily  escapes 
by  pushing  the  plug  of  lanolin  into  the  upper  non-constricted  portion  of  the 
tube. 

2.  When  required  for  use,  melt  the  layer  of  lanolin  in  the  flame  (it  liquefies 
at  42°  C.),  and  sow  the  organism  in  the  ordinary  way  through  the  melted 
lanolin.  Cool  rapidly  to  solidify  the  fat.  Growth  takes  place  in  what 
is  practically  a  sealed  tube  (Rosenthal). 

L.  Tarozzi's  method. — To  grow  the  strictly  anaerobic  organisms  (Bacillus 
tetani,  Bacillus  maligni  cedematis,  etc.)  by  this  method  it  is  only  necessary 
to  add  to  broth  contained  in  ordinary  tubes  a  fragment  of  tissue  freshly 
removed  from  a  rabbit,  mouse  or  guinea-pig,  and  to  proceed  as  in  the  case  of 
aerobic  organisms. 

Pieces  of  liver,  spleen,  kidney  or  lymphatic  glands  may  be  used  with  success, 
but  blood,  milk,  or  the  connective  tissues  are  useless  for  the  purpose.  To 
tubes  of  broth  add  a  small  piece  of  one  of  the  above-mentioned  internal 
organs  which  has  recently  been  excised  with  the  usual  aseptic  precautions. 
Incubate  the  tubes  for  a  day  or  two  at  37°  C.,  and  they  are  then  ready  for 
use.  They  may  be  heated  to  100°-107°  C.  for  a  minute  or  two,  but  if  the 
heating  be  prolonged  for  more  than  5  minutes  growth  will  fail.  Cultures 
will  grow  even  if  the  piece  of  tissue  be  removed  before  sowing. 

A  number  of  other  substances  have  a  similar  action  in  facilitating  the  growth 
of  anaerobic  organisms.  Wrzosek,  Ori,  for  example,  were  able  to  obtain  cultures 
under  ordinary  conditions  in  broth  by  simply  adding  pieces  of  vegetable  tissue 
(potato,  elder  pith,  mushrooms,  etc.)  to  the  medium.  Tarozzi  used  a  slightly 
alkaline  glucose-broth,  which  had  been  heated  under  a  pressure  of  two  atmospheres 
in  the  autoclave,  with  successful  results.  Aperlo  was  able  to  grow  strictly  anaerobic 
organisms  in  a  simple  pep  tone- broth  by  sterilizing  the  medium  for  half  an  hour 
under  a  pressure  of  half  an  atmosphere,  and  using  it  within  24  hours  of  its 
preparation. 

Kata  also  succeeded  with  ordinary  broth  containing  a  small  piece  of  agar  and 
0'3-0'7  per  cent,  of  sodium  sulphite,  and  even  better  with  the  same  amount  of 
sulphite  and  a  little  fresh  serum.  The  latter  medium  would  appear  to  be  very 
useful  for  toxin  production. 

Pf uhl  recommends  a  broth  made  with  liver  instead  of  ordinary  meat,  and  sterilized 
in  the  autoclave.  Satisfactory  results  were  also  obtained  with  the  following 
technique :  To  a  tube  containing  10  c.c.  of  ordinary  broth  add  1  gram  of  spongy 
platinum,  boil  for  10  minutes,  sow  as  soon  as  cool  and  put  in  the  incubator  without 
shaking  the  tube. 

The  vitality  of  anaerobic  organisms  is  exhausted  much  more  quickly  on 
media  prepared  on  these  principles  than  on  media  under  anaerobic  conditions 
(Jungano  and  Distaso). 


CULTIVATION   ON  SOLID  MEDIA  99 

2.  Solid  media. 

(i)   Stab  cultures. 

A.  In  test-tubes.  Method  recommended,  (a)  Gelatin.—  1.  Heat  a  tube  of 
sterile  gelatin  to  boiling,  taking  care  not  to  let  the  medium  froth  and  boil 
over.  Boil  for  several  minutes.  [This  is  best  done  in  a  water  bath.] 

A  few  drops  of  sodium  sulphindigotate  solution  may  be  added  to  the  gelatin 
before  boiling  it  and  if  this  be  done  the  medium  will  be  decolourized  by  the  growth 
of  the  organism. 

2.  Cool  the  gelatin  rapidly,  and  when  it  is  set  sow  a  stab  cul- 
ture with  a  fine  platinum  wire. 

A  little  air  would  ordinarily  be  introduced  with  the  needle,  and  the 
following  arrangement  is  devised  to  obviate  this.  Mount  the  wire  on 
the  wall  of  a  piece  of  glass  tubing,  and  connect  the  other  end  of  the 
latter  to  a  hydrogen-generating  apparatus  by  means  of  a  piece  of  india- 
rubber  tubing  (fig.  89).  To  use  the  needle,  after  flaming  it,  take  up 
the  material  to  be  sown,  then  turn  on  the  hydrogen  and  sow  in  a  current 
of  the  gas.  In  this  way  the  oxygen  of  the  atmosphere  is  prevented  from 
reaching  the  needle  track. 

3.  After  sowing,  dip  the  gelatin  tube  into  very  cold  water  and 
pour  a  layer  of  agar  over   the    surface  with  a  Pasteur   pipette. 
Replace  the  plug.     The  object  of  this  procedure  is  to  form  a  plug 
impervious  to  air  on  top  of  the  gelatin.     Sterilized  oil  or  liquid 
vaseline,  etc.,  may  be  used  instead  of  agar. 

Note.  —  The  agar  plug  may  be  omitted  if  some  very  oxidizable 
substance  capable  of  absorbing  oxygen  be  added  to  the  culture 
medium  (Liborius,  Kitasato).     The  best  substances  for  the  pur- 
pose are  glucose  (2  per  cent.),  sulphindigotate  of  sodium  (Crl  per 
cent.),  sodium  formate  (O5  per  cent.). 

Liborius  recommends  the  following  medium  :  wf  re  8f  b~r 

Ordinary  agar,    .......         1000  grams.  sowing    an- 

S 


. 
bodium  sulphindigotate,       -  1  gram. 

Nearly  fill  the  tubes  with  the  medium,  and  sow  deep  stab  cultures  as 
described  above. 

(b)  Agar.  —  The  method  is  the  same  as  in  the  case  of  gelatin. 

B.  Absorption  of  oxygen  by  an  aerobic  organism  (Roux).  —  Proceed  as 
above,  and  when  the  agar  plug  has  set  sow  the  surface  with  B.  subtilis.     This 
organism  is  strictly  aerobic   and  absorbs  the  oxygen  present  in  the  tube, 
while  growth  below  takes  place  under  anaerobic  conditions  in  an  atmosphere 
free  from  oxygen. 

To  reach  the  anaerobic  organism  without  contaminating  it  with  the  B.  subtilis, 
wash  the  outside  of  the  tube  with  perchloride  of  mercury,  cut  it  across  about  the 
level  of  the  middle  of  the  growth,  break  off  the  lower  part  of  the  tube,  and  the 
anaerobic  organism  can  then  be  removed  without  contaminating  it. 

C.  Roux's  pipette.  —  1.  Flame  and  break  off  the  point  of  a  Roux's  pipette. 
Dip  the  end  into  a  tube  of  sterile  gelatin  which  has  just  been  boiled.     Draw 
the  gelatin  into  the  tube  until  it  reaches  the  constriction  a,  fig.  67,  p.  75. 
Seal  the  narrow  end  of  the  pipette  and  the  upper  end  at  the  constriction. 
Dip  the  whole  tube  into  cold  water  to  cool  it  quickly. 

2.  When  the  gelatin  has  set,  pass  the  upper  part  rapidly  through  the 
flame,  and  then  break  off  the  point  a  with  a  pair  of  forceps.  Through  the 
opening  sow  a  stab  culture  with  a  fine  wire.  Seal  the  opening  in  a  flame. 


100     CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 


a 


-FlG    9Q  _ 

Boux's'method 
of  growing  an- 

isms. 


3.  To  open  the  tube  when  growth  has  taken  place,  break  off  the  lower  end 
over  a  sterile  glass  plate.  If  the  upper  end  were  opened  first  the  pressure 
of  the  gases  formed  during  the  growth  of  the  organism  would  be  sufficient 
to  forcibly  expel  the  contents  of  the  tube. 

D.  Hydrogen  method  (Roux).  —  This  method  is  more  difficult  than 
those  just  described. 

1.  Take  a  tube  of  sterile  gelatin,  and  constrict  in  the  blow-pipe 
just  below  the  plug  (a,  fig.  90). 

2.  Select  a  sterile  Pasteur  pipette  the  smaller  end  of  which  will 
easily  pass  through  the  constriction,  and  bend  it  at  a  right  angle  below 
the  wool   plug.     Connect   the   plugged   end   of   the   pipette   with  a 
hydrogen  apparatus. 

3.  Melt  the  gelatin  in  a  water  bath.     Flame  the  narrow  part  of 
the  pipette  to  sterilize  it,  and  after  breaking  off  the  point  pass  it 
between  the  wool  and  the  side  of  the  tube  down  to  the  bottom  of 
the  gelatin. 

4.  Pass  a  stream  of  hydrogen  through  the  medium  for  some  minutes, 
and  then  withdraw  the  pipette  a  little  so  that  the  hydrogen  passes 
over  the  surface  of  the  gelatin  and  prevents  air  gaining  access  to  the 
medium  while  it  is  being  cooled. 

5.  Take  out  the  wool  plug  and  sow  a  stab  culture  with  a  fine  wire, 
fae  current  of  hydrogen  being  maintained  meanwhile. 

6.  When  the  tube  is  sown,  take  out  the  pipette  and  seal  the  tube 
as  quickly  as  possible  at  the  constricted  part  a. 

[E.  Bulloch  s  apparatus  can  be  used  equally  well  with  solid  as  with  liquid 
media  (p.  96).] 

(ii)   Surface  cultures. 

Gelatin  and  agar. 

|  Bulloch  s  apparatus  is  available  for  the  growth  of  anae- 
Tobic  organisms  in  surface  culture  (pp.  96  and  102).] 

Roux  s  tube.  —  Roux's  tube  for  stroke  cultures  of  anaerobic 
organisms  consists  of  an  ordinary  test-tube  T  drawn  out  above 
(A)  and  provided  with  a  lateral  branch  B  (fig.  91). 

1.  Pour  some  gelatin  into  the  lower,  wider  part  of  the 
tube  T,  using  a  narrow-stemmed  funnel  for  the  purpose. 
Seal  the  tube  at  the  constriction  a  in  its  upper  part.     Plug 
the  side  tube  B  with  wool  between  the  two  constrictions  b 
and  b'.     Sterilize  in  the  autoclave. 

2.  Attach  B  to  the  water  pump,  stand  the  tube  in  a  water 
bath  at  a  temperature  just  sufficient  to  keep  the  gelatin 
melted  while  the  tube  is  exhausted  and  washed  two  or  three 
times  with  hydrogen. 

3.  When  the  air  has  been  displaced  by  hydrogen,  leave  the 
tube  in  a  slanting  position  while  the  gelatin  sets. 

4.  Then  flame  the  upper  part  of  A,  break  off  the  point  a, 
and  sow  a  stroke  culture  through  the  opening  ;  the  tube  B 
must  at  the  same  time  be  connected  with  the  hydrogen- 
generating  apparatus   and   a    stream   of   hydrogen   passed 

into  the  tube  to  prevent  the  access  of  air.     Seal  the  top  of  the  tube  A 
again. 

5.  It  now  only  remains  to  seal  B  at  the  constriction  b'.     Growth  then 
takes  place  in  an  atmosphere  of  hydrogen.     If  necessary  the  tube  can  be 
again  exhausted  after  sowing  and  sealing  a,  before  sealing  b'. 


B 


FIG.  91.— Roux's 
tube  for  stroke  cul- 
tures of  anaerobic 
organisms. 


METHODS   OF  ISOLATION 


101 


Potato. 


Blow   on   to   an   ordinary  potato-tube   below  the  con- 
tube  B,   and  plug   the  latter  with  wool  between  two 


b  B  b, 


Roux's  tube. — 1. 

striction,   a  lateral 

constrictions  (fig.  92).  (These  tubes  can 
be  bought  ready  made.)  Place  a  piece 
of  potato  in  the  tube,  and  sterilize  it  in 
the  autoclave  at  120°  C. 

2.  Sow  the  potato  in  the  ordinary  way, 
and  then  seal  the  upper  end  of  the  tube 
below  the  wool  plug  in  the  flame  (fig.  92). 

3.  Attach  B  to  the  pump.     Exhaust 
and  wash  with  hydrogen. 

4.  Seal  the  side  tube  B  at  the  constric- 
tion b'  under  a  vacuum,  and  incubate. 


SECTION  HI.— THE  ISOLATION  OF 

ANAEROBIC  ORGANISMS. 

1.  Plate  method. 

A.  On  sheets  of  glass. — The  method  is 
similar   to   that    described   for   aerobic 
organisms.     The   technique   is   difficult 
but  has  the  advantage  that  the  colonies 

r>nn       VIA       AYarmnorl       nnrlor      +lia      rmVrn        FlG.  92. — Roux's  tubes  for  sowing  cultures  Of 

can     be    examined    under    tne    micro-  anaerobic  organisms  on  potato, 

scope. 

1. — (1)  Sow  three  tubes  of  liquefied  sterile  gelatin  with  the  organism  under  investiga- 
tion, and  pour  three  plates  as  in  isolating  aerobes  (p.  78). 

(2)  Have  a  vacuum  desiccator  (previously  washed  inside  with  perchloride)  at  hand,  and 
pour  some  potassium  pyrogallate  (p.  89)  into  the  sulphuric  acid  vessel.     A  vacuum 
incubator  (p.  104)  can  also  be  used. 

(3)  Arrange  the  plates  on  the  shelves  as  they  are  poured. 

(4)  Lute  the  bell  jar,  exhaust,  and  wash  with  hydrogen.     Disconnect  the  bell  jar  by 
closing  the  tap  connecting  it  to  the  pump. 

2.  Turro  has  simplified  the  method  by  arranging  the  plates  on  glass  benches  in  a  large 
glass  dish  into  the  bottom  of  which  some  potassium  pyrogallate  is  poured.     The  ground 
glass  cover  of  the  glass  dish  is  then  sealed  with  paraffin.     Agar  can  be  used  for  the  plates, 
and  the  whole  incubated. 

B.  Kitasato's  dish. — A  circular  flat  glass  dish  of  the  size  of  a  Petri  dish  is  fitted 

with  two  tubes  A  and  B  on  opposite  sides. 
The  tube  B  is  drawn  out  and  sealed.  A  is 
plugged  with  wool  (fig.  93). 

This  is  a  satisfactory  though  fragile  and 
rather  expensive  piece  of  apparatus. 

1.  Sterilize  the  apparatus  in  the  hot  air 
sterilizer. 

2.  After  flaming  the  end  of  B,  break  off  the 

point   and  dip   the  end   into   a  gelatin  tube    already  sown,  and   aspirate  the  medium 
into  the  dish  through  A.     Seal  B,  and  let  the  gelatin  solidify. 

3.  Attach   A   to   the   pump.     Exhaust,   and   wash   with   hydrogen.     Seal   A  at  the 
constriction  a. 

C.  Bombicci's  apparatus. — This  vessel  is  cheaper  than  Kitasato's.     It  consists 
of  a  circular  flat  glass  dish  with  a  cylindrical  appendix  of  about  10  c.c.  capacity. 

1.  Pour  the  medium,  agar  or  gelatin,  into  the  appendix,  plug  the  neck  with  wool 
and  sterilize  in  the  autoclave. 

2.  Select  an  india-rubber  plug  with  two  holes  which  fits  the  neck  of  the  dish.     Fit  it 
with  two  tubes  as  shown  in  the  figure  and  plug  the  horizontal  limb  of  each  with  wool 


FIG.  93. — Kitasato's  dish. 


102      CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 


between  two  constrictions.     Wrap  the  plug  with  the  tubes  in  position  in  paper,  and 
sterilize  separately  from  but  at  the  same  time  as  the  dish. 

3.  Keep  the  gelatin   or  agar  liquefied  in  a  water  bath  at 
30°-40°  C.  as  the  case  may  be,  while  sowing  the  medium.     Take 
the  india-rubber  plug  out  of  its  wrapper  and  fit  it  into  the  neck 
of  the  dish  as  quickly  as  possible. 

4.  Lute  the  plug  with  Golaz's  wax,  attach  A  to  the  hydro- 
gen-generating apparatus,  keeping  the  medium  liquefied  in  the 
water  bath,  and  pass  a  stream  of  hydrogen  through  the  medium 
for  a  few  minutes.     Lay  the  apparatus    horizontally   so   that 
the  medium  flows  into  the  dish  and  continue  the  current  of  hy- 
drogen for  several  minutes.     Exhaustion  may  be  combined  with 
washing  with  hydrogen  if  it  be  thought  necessary. 

5.  Seal  the  ends  of  A  and  B  beyond  the  wool  plugs. 

D.  Zinsser's  method. — Zinsser  uses  an  apparatus 
similar  to  a  Petri  dish,  but  deeper,  and  having  an  an- 
nular space  of  5-6  mm.  between  the  dish  and  the  cover. 
The  agar  or  gelatin,  as  the  case  may  be,  is  sown  and 
poured  into  the  dish,  and  after  it  is  set  is  inverted  on 
to  the  cover,  into  which  a  little  alkaline  pyrogallol  is 
poured  (p.  89).  A  layer  of  oil  is  poured  on  the  surface 
FIG.  94.-Bombicci's  dish.  of  tne  pyrogallate  in  the  annular  space. 

E.  Tarozzi  s  method. — Tarozzi  uses  an  alkaline  glucose-  agar  which  has 
been  heated  under  a  pressure  of  two  atmospheres  (p.  98).     The  medium  is 
poured  to  a  depth  of  1  cm.  into  Petri  dishes  with  ground-glass  covers,  which 
are  luted  with  paraffin. 

F.  Marino's  method. — 1.  Take  a  number  of  Petri  dishes,  remove  the  lids, 
and  place  the  dishes,  cavity  upwards,  over  (and  therefore  partly  within) 
them.     Wrap  in  paper  and  sterilize  in  the  hot  air  sterilizer. 

2.  Take  a   number  of  large  test-tubes,   and  into   each   pour  30  c.c.   of 
O5  per  cent,  glucose-agar.     Sterilize  in  the  autoclave. 

3.  Cool  the  agar  to  40°-42°  C.  in  a  water  bath.     Add  to  the  contents  of 
each  tube  1  c.c.  of  rabbit-  or  horse-serum  previously  heated  at  55°  C.  for 
20  minutes. 

4.  Sow  the  tubes  by  the  dilution  method. 

5.  Pour  the  contents  of  each  tube  into  the  lid  of  one  of  the  sterile  Petri 
dishes,  and  cover  with  the  other  part  of  the  dish  in  such  a  way  that  the  agar 
is  contained  between  and  compressed  by  two  sterile  glass  surfaces,  the  cavity 
•of  the  dish  being  obviously  upwards.     Cover  with  a  sterile  glass  plate  large 
enough  to  project  beyond  the  edges  of  the  Petri  dish  to  protect  it  from 
contamination.     Incubate. 

6.  After  the  colonies  have  grown,  gently  separate  the  agar  from  one  of  the 
glass  surfaces,  leaving  it  adhering  to  the  other,  and  pick  of!  with  a  fine- 
pointed  pipette,  any  colonies  it  is  thought  desirable  to  examine. 

When  separating  the  medium  from  one  of  the  glass  surfaces  it  often  happens  that 
the  agar  is  torn  ;  so  it  may  be  that  the  colony  which  was  wanted  cannot  be  found, 
or  else  that  it  has  become  contaminated,  by  rubbing  up  against  another  colony  or 
by  contact  with  the  water  of  condensation. 

Lief  man,  Fehrs  and  Sachs-Miike's  modification  of  Marino's  method  obviates  this 
defect.  Instead  of  the  lower  part  of  the  Petri  dish  a  plate  of  sterile  glass  is  used 
as  a  cover,  and  sufficient  medium  is  poured  into  the  lid  to  slightly  overflow  the 
edges.  In  covering  with  the  sheet  of  glass  care  must  be  taken  that  no  air  bubbles 
are  included. 

[G.  Bulloch's  apparatus.  The  technique  as  now  generally  adopted  has 
been  explained  at  p.  96.  The  only  modification  required  in  the  present 


METHODS   OF  ISOLATION 


103 


connexion  is  the  use  of  Petri  dishes  containing  a  solid  medium  instead  of 
tubes  of  a  liquid  medium.  It  will  be  necessary,  of  course,  to  have  a  glass 
tripod  or  a  thick  sheet  of  cork  on  which  to  stand  the  dishes,  in  order  to 
prevent  the  lower  ones  being  flooded  with  the  pyrogallate  solution.  It  is 
advisable  also  to  stand  some  water  in  a  Petri  dish  on  the  top  of  the  upper- 
most plate.] 

2.  Tube  method. 

A.  Esmarch's  tubes. — Frsenkel,   Roux  have  adapted  the  Esmarch  tube 
method   of  isolating   aerobic   organisms   to   the   isolation   of 

anaerobic    species.     The    technique    recommended    by   these 
authors  is  somewhat  complicated,  and  is  now  very  rarely  used. 

Frsenkel's  method. — Fraenkel  prepares  an  Esmarch  tube  (p.  81), 
and  after  sowing  the  medium  in  air,  displaces  the  latter  by  hydrogen 
by  means  of  an  arrangement  similar  to  that  described  in  Bombicci's 
method  (p.  101).  After  passing  the  hydrogen  through  the  medium 
for  5  or  10  minutes  the  tube  is  rolled  as  in  the  ordinary  Esmarch 
method. 

Roux's  method. — Roux  sterilizes  the  medium  in  a  test-tube  the 
upper  part  of  which  has  been  narrowed  by  drawing  it  out  in  the 
flame  (fig.  95,  left-hand  figure).  When  cool  but  still  liquid  the 
medium  is  sown.  The  narrowed  part  of  the  tube  is  then  constricted 
at  two  points  and  the  wool  plug  pushed  down  between  them  (fig. 
95,  right-hand  figure.)  Attach  the  tube  to  a  water  pump,  exhaust 
and  wash  with  hydrogen,  seal  at  the  upper  constriction,  and  roll  FlGh'95't~bES" 
the  gelatin.  To  remove  the  colonies,  cut  off  the  upper  part  of  the  applied  to  anae- 
tube  and  pass  a  platinum  wire  through  the  opening.  robic  cultivation. 

B.  Vignal's  tube.    Method  recommended. — 1.  Take  a  piece  of  glass  tubing 
about  1  metre  long  and  3  or  4  mm.  in  diameter.     Draw  out  one  end  in  the 
flame  and  plug  the  other  with  wool.     Make  a  constriction  in  the  tube  3  or 
4  cm.  below  the  wool  plug  (fig.  96).     Heat  the  tube  thoroughly  in  the  flame 

to  sterilize  it. 

2.  Heat  a  tube  of  sterile  gelatin  to  boiling  point  (the  medium 
may,  if  desired,  be  coloured  with  sulphindigotate  of  sodium). 
Let  the  gelatin  cool  in  a  current  of  hydrogen  (p.  100),  but  sow  it 
before  it  sets,  also  in  a  current  of  hydrogen.     Rotate  the  tube 
between   the   hands   to   distribute   the    organisms   through   the 
medium. 

3.  Flame  the  sealed  end  of  the  tube,  and  break  off  the  point. 
Dip  the  end  into  the  gelatin,  and  aspirate  the  medium  into  the 
tube  up  to  the  constriction  A.     (It  is  necessary  to  take  care  that 
no  bubbles  of  gas  enter  the  tube.)     Seal  the  pointed  end  and 
then  close  the  tube  at  A  (fig.  96,  A'). 

Colonies  soon  appear  scattered  through  the  gelatin.  The 
growth  can  be  removed  by  carefully  flaming  the  tube  or  washing 
it,  first  in  perchloride  then  in  alcohol,  in  the  neighbourhood  of  the 
colony  which  it  is  desired  to  examine.  Cut  the  tube  at  the  steri- 
lized part  and  remove  the  growth  with  a  needle. 

C.  Method    of   Liborius-Veillon.    Method    recommended.— Liborius'    agar 
(p.  99)  which  is  used  for  deep  stab  culture  is  also  available  for  the  isolation 
of  anaerobic  micro-organisms.     The  tubes  are  sown  by  the  dilution  method 
(p.  79)  and  cooled  rapidly.     For  the  examination  and  sub-cultivation  of  the 
colonies,  Liborius  recommended  turning  out  the  agar  on  to  the  inside  of  the 
lid  of  a  sterile  Petri  dish  and  cutting  out  the  colonies  with  a  sterile  knife, 
a  process  which  was  not  only  rather  difficult  but  exposed  the  colonies  to 


FIG.  96  — 
Vignal's  tube. 


104      CULTIVATION   OF  ANAEROBIC  MICRO-ORGANISMS 

contamination.  To  overcome  these  disadvantages,  the  method  has  been 
modified  by  Veillon  and  as  modified  by  him  is  now  [one  of]  the  best 
methods  of  isolating  anaerobic  organisms. 

1.  Fill  a  number  of  large  test-tubes  (22  cm.  x  15  mm.)  to  a  depth  of  10  to 
15  cm.  with  some  quite  transparent  agar  containing  1*5  per  cent,  of  glucose. 
Sterilize  in  the  ordinary  way  but  do  not  allow  the  temperature  to  exceed 
120°  C. 

2.  When  ready  to  sow  the  tubes,  heat  five  to  ten  of  them  to  100°  C.  in  a 
water  bath,  and  boil  them  for  20  minutes  or  so  to  liquefy  the  agar  and  drive 
off  the  air  dissolved  in  the  medium.     Then  transfer  the  tubes  to  a  water  bath 
at  40°  C.  to  keep  the  agar  liquid  until  sown. 

3.  Add  one  drop  of  the  material  to  be  sown  to  the  first  tube,  and  disseminate 
it  by  rolling  the  tube  between  the  hands. 

4.  Sow  the  second  tube  with  a  few  drops  from  the  first,  the  third  from  the 
second  and  so  on,  as  previously  described. 

5.  Immediately  the  tubes  are  sown,   cool  them  rapidly  in  the  upright 
position.     Incubate. 

Aerobic  organisms  grow  in  the  upper  part  of  the  medium  which  contains 
a  certain  amount  of  air  in  solution,  while  the  anaerobes  multiply  in  the 
deeper  layer. 

6.  When  carefully  examined  it  will  be  found  that  growth  soon  makes  its 
appearance,   the  number  of  colonies  depending  upon  the  extent  to  which 
the    material    with    which    they    were    sown    was    diluted.      Examine    the 
different    colonies   with   the   naked   eye   and   with   a   lens   and   select   the 
tubes  containing  the  smallest  number  of  colonies  for  the  purposes  of  sub- 
cultivation. 

To  sub-cultivate,  take  a  Pasteur  pipette  with  a  fine  point,  break  off  the  end, 
and  holding  the  culture-tube  horizontally  remove  the  wool  plug  and  pass 
the  fine  end  of  the  pipette  into  the  agar  towards  the  colony  to  be  removed  : 
as  the  pipette  is  passed  through  the  colony  some  of  the  growth  is  forced 
into  it ;  withdraw  the  pipette  and  sow  the  colony  in  a  fresh  tube  of 
medium. 

It  facilitates  the  process  of  sub-cultivation  to  put,  as  Guillemot  advises,  an  india- 
rubber  teat  on  the  plugged  end  of  the  pipette  ;  the  colony  can  then  be  more  easily 
drawn  into  the  pipette  by  aspiration,  and  forced  out  into  the  new  medium  by 
compression  of  the  teat. 

Great  care  must  be  taken  that  the  pipette  does  not  touch  any  colony  other 
than  that  to  be  sub -cultivated ;  the  only  way  of  avoiding  such  an  accident 
is  to  work  with  cultures  in  which  the  colonies  are  few  in  number  and  suffi- 
ciently well  isolated  one  from  another. 

Note. — It  is  often  an  advantage  to  use  a  medium  containing  serum,  since  many  anaerobic 
bacteria  grow  better  in  albuminous  media.  Prepare  the  agar  as  above,  melt  the  contents 
of  the  tubes,  and  cool  to  40°  C.  in  a  water  bath,  then  to  each  tube  for  two  parts  of  agar 
add  one  part  of  sterile  liquid  serum  also  heated  to  40°  C.  :  mix  the  agar  and  serum 
thoroughly,  keeping  the  medium  at  40°  C.  to  prevent  the  contents  solidifying,  and  sow 
with  the  material. 


SECTION  IV.— VACUUM  INCUBATORS. 

Anaerobic  organisms  can  be  cultivated  in  ordinary  culture  vessels,  provided 
that  these  are  incubated  in  a  special  form  of  incubator  which  can  be  exhausted 
and  the  vacuum  maintained.  A  little  water,  or,  better,  solution  of  potassium 
pyrogallate  which  absorbs  oxygen,  should  always  be  placed  in  these 
incubators  to  prevent  desiccation  of  the  medium. 

In  discussing  isolation  of  anaerobic  organisms  by  the  plate  method,  Roux's 


VACUUM  INCUBATORS  105 

vacuum  bell  jar  has  already  been  described.     Tretrdp's  (fig.  97),  or  Baginski's- 
apparatus,  or  Adnet's  vacuum  incubator  are  also  available.     The  last  is  a 


FIG.  97. — Tretrop's  apparatus,  in  which  to  grow  anaerobic  cultures. 

stout-walled  incubator  which  can  be  hermetically  closed  by  means  of  a  door 
with  an  india-rubber  washer  and  is  provided  with  a  Roux's  regulator  and  a 


gas  burner. 


CHAPTER  VII. 
THE  MICROSCOPE. 

Introduction. 

Section  I. — The  microscope  stand,  p.  106. 

Section  II. — The  optical  parts  of  the  microscope,  p.  107. 

A.  The  objectives,  p.  107.     B.  The  eyepieces,  p.  116. 
Section  III. — The  care  of  the  microscope,  p.  117. 
Section  IV. — The  method  of  using  the  microscope,  p.  118. 
Section  V. — The  measurement  of  microscopical  objects,  p.  121. 

1.  The  experimental  determination  of  the  magnification  produced  by  a  system  of 
lenses,  p.  121.     2.  The  measurements  of  objects  under  the  microscope,  p.  122. 
Section  VI. — Dark-ground  illumination,  p.  123. 

1.  The  application  of  dark-ground  illumination  to  micro-biology,  p.  124.  2.  The 
construction  of  the  dark-ground  illuminator,  p.  124.  3.  The  method  of  using  the 
instrument,  p.  125. 

FOR  bacteriological  work  a  good  microscope,  which  will  magnify  from  600  to 
1200  diameters,  is  necessary.  It  is  seldom  that  a  higher  magnification  than 
1200  diameters  is  required,  though  for  a  few  micro-organisms,  e.g.  the  organism 
of  pleuro-pneumonia,  a  magnification  of  2000  diameters  may  be  useful.  It 
is  to  be  remembered  however  that  with  the  very  best  instruments  it  is 
impossible  to  see  organisms  measuring  less  than  0*0001  mm.  (0*1  //.)  in  diameter 
(p.  113). 

A  microscope  may  for  purposes  of  description  be  regarded  as  consisting 
of  two  parts,  the  mechanical  (the  microscope  stand)  and  the  optical  (the 
lenses)  portions  respectively. 

SECTION   I.— THE   MICROSCOPE   STAND. 

The  stand  of  the  microscope  must  be  firm  and  rigid,  and  it  is  desirable 
that  the  base  be  hinged  to  the  body  so  that  the  latter  can  be  tilted.  The 
tube  should  have  a  rack  and  pinion  mechanism  and  a  micrometer  screw 
adjustment  for  the  grosser  and  more  delicate  movements  respectively  in 
focussing.  The  stage,  of  ebonite  or  metal,  should  be  large,  and  it  is  an  advan- 
tage if  it  can  be  centred  and  mechanically  moved.  The  mirror,  by  means  of 
which  the  light  is  transmitted  to  the  object,  should  be  concave  on  one  side 
and  flat  on  the  other.  The  stand,  moreover,  should  be  so  constructed  that 
an  Abbe  condenser  can  be  fitted  below  the  stage.  A  diaphragm  either  of  the 
cylindrical  or  iris  pattern  is  also  essential.  It  will  be  found  a  great  advantage 
to  have  a  triple  nosepiece  capable  of  carrying  three  objectives,  so  that  one 
lens  can  be  readily  and  quickly  substituted  for  another. 


MAGNIFICATION 


107 


SECTION    II.— THE    OPTICAL  PARTS 
OF  THE  MICROSCOPE. 

The  great  difficulty  in  selecting  a 
microscope  is  the  choice  of  the  lenses. 
For  ordinary  work  two  eyepieces,  and 
four  objectives  including  a  TVin.  oil- 
immersion  lens,  are  all  that  is  neces- 
sary.1 

A  TVm-  or  TVm-  homogeneous  im- 
mersion lens  may  be  of  use  occasionally. 

In  addition  to  the  microscope  and 
its  lenses,  it  is  convenient  to  have  a 
camera  lucida  and  a  stage  and  ocular 
micrometer. 

A.  The  objectives.2 

The  use  of  a  microscope  is  to  magnify 
the  details  of  an  object,  so  that  those 
invisible  to  the  naked  eye  may  with  its 
aid  be  easily  seen.  The  essential  re- 
quisites then  in  good  lenses  are  definition 
and  magnification. 


1.  Magnification. 

The  apparent  linear  size  of  an  object  AB 
(fig.  99)  varies  inversely  as  its  distance  BK 
from  the  eye  of  the  observer,  and  depends 
upon  the  tangent  of  the  visual  angle  a  which  it  subtends  at  the  nodal  point  K 
of  the  eye.  T>  \ 

tan  a  =  tan  BK  A  =  ^^' 
JVr> 

Now,  let  B'K  denote  the  least  distance  of  distinct  vision  (10  inches),  and  let  it  be 
denoted  by  I. 


FIG.  98. — A  microscope. 


Then  the  greatest  apparent  size  of  BA  to  the  unaided  eye  is  when  it  is  in  the 
position  B'A',  and  its  apparent  size  is  then 

,    B'A'     B'A' 
=  KB'=^T' 

It  follows  from  this  that  the  larger  the  angle  subtended  by  the  object  at  K,  the 
larger  will  the  object  appear  to  be.  And  a  microscope  is  nothing  more  than  an 
instrument  with  which  to  increase  the  size  of  this  angle. 

1  In  French  makes,  and  also  in  Reichert's  and  Leitz'  lenses  a  No.  I.  or  No.  II.  and  a 
No.  III.  eyepiece,  and  a  2,  6,  and  8  or  9  dry  objective.  In  Zeiss'  list  the  corresponding 
objectives  are  AA,  DD,  and  E  in  the  dry  series,  and  eyepieces,  2,  4  and  8. 

[ 2  The  remainder  of  this  section,  dealing  with  the  theory  of  the  microscope,  has  been 
rewritten  and  considerably  extended. — H.  J.  H.] 


108 


THEORY   OF  THE   MICROSCOPE 


If  a  convex  lens  of  focal  length  2  in.  be  placed  2  in.  in  front  of  an  object  AB 
(fig.  100),  the  divergent  pencil  of  light  from  A,  HAO,  will  emerge  from  the  lens  as  a 
parallel  beam  F"H,  LO,  as  if  it  came  from  an  object  A'  at  an  infinite  distance  off, 
while  the  divergent  pencil  of  rays  from  B  will  emerge  from  the  lens  as  a  beam  parallel 
to  the  axis,  as  if  it  came  from  B'  at  an  infinite  distance  off.  The  image  is  virtual, 


FIG.  100. 


not  real,  and  cannot  therefore  be  received  on  a  ground-glass  screen,  but  it  appears 
to  the  eye  as  if  it  came  from  a  big  object  at  an  enormous  distance  away. 

The  visual  angle  which  this  huge  image  at  an  infinite  distance  subtends  at  the 
eye  will  obviously  be  BOA  or  0',  and 

BA        BA 
"-/'' 


tan  #'  =  TS^,= 


OB      - 


But 


tan  0  —  — f  ; 


tan 


I      10 


The  object  is  therefore  magnified  5  times  by  the  convex  lens  of  2  in.  focal  length 
placed  2  in.  from  it.  The  positive  sign  shows  that  the  image  is  erect  and  virtual. 

Now  suppose  it  be  required  to  magnify  an  object  10  times  (linear)  it  is  clear 
that  a  lens  of  1  in.  focal  length  would  have  to  be  used,  for 

10      10 


and  to  obtain  a  magnification  of  400  it  would  be  necessary  to  have  a  lens  of  focal 
length  j\j  in.,  and  the  object  would  therefore  have  to  be  not  more  than  ^  in.  away 
from  the  lens  ;  this  in  most  cases  would  be  impossible,  not  to  speak  of  the  extreme 
spherical  and  chromatic  aberration  that  would  be  induced  by  using  a  single  lens  of 
that  high  degree  of  curvature. 

There  is  a  simple  method  by  which  some  of  these  defects  may  be  overcome,  which 
may  be  illustrated  by  a  consideration  of  the  simple  magnifying  glass  of  1  in.  focal 
length  mentioned  above.  In  this  case  the  lens  must  be  placed  1  in.  away  from 
the  object  to  induce  a  magnification  of  ten  diameters.  Now  if  this  biconvex 
lens  be  split  down  the  middle,  two  plano-convex  lenses  will  be  formed  each  of  2  in. 
focal  length.  On  placing  one  of  these  lenses  2£  in.  away  from  the  object  AB,  an 
enlarged  inverted  image  ab  will  be  formed  at  a  distance  of  18  in.  from  the  lens 
(fig.  101). 

(For      H4;    ':H~f^~r-is;    ••?=-18in- 

where  p  is  the  distance  from  the  object  and  q  the  distance  from  the  image  to  the 
lens,  and  /  the  focal  length  of  the  lens.  ) 

On  now  placing  the  second  plano-convex  lens  2  in.  beyond  this  image  (i.e. 
with  the  image  at  its  focal  distance,  it  will  be  magnified  again.  This  is  the  funda- 


THE   COMPOUND  MICROSCOPE 


109 


mental  principle  of  the  compound  microscope.     The  first  lens,  or  objective,  forms 
an  inverted  image  8  times  the  size  of  the  object. 

IH_F'I         F'l  /'  2 


(For 


i 
-  or 


=  -8. 


o        BA~BA~F'B~F'I-BI~/'-P~2-2i 

The  negative  sign  shows  that  the  image  is  inverted.) 

The  second  lens  or  eyepiece  is  now  placed  2  in.  from  this  image,  i.e.  20  in.  from 


FIG.  101. 


the  objective,  so  that  the  inverted  image  ba  is  in  its  first  focal  plane.  Consequently 
the  image  ba  will  be  seen  under  a  magnification  of  ^  =-^  or  5.  The  total  magnifica- 
tion will  be  therefore  -  8  x  -^  =  -  40. 

It  follows,  then,  that  by  this  arrangement  the  working  distance  is  increased  from 
1  in.  to  2£  in.,  that  the  magnification  is  increased  from  10  to  -40,  while  the 
errors  from  spherical  and  chromatic  aberration  are  rather  less  than  with  a  single 
biconvex  lens  of  1  in.  focus.  Indeed,  with  the  compound  instrument,  what  is 
called  "  pincushion  distortion  "  would  be  almost  entirely  obviated.  If  an  object 
were  a  network  of  squares,  it  would  be  found  that,  on  using  a  simple  magnifying 
lens,  it  would  present  the  appearance  shown  in  fig.  102  (pincushion  distortion), 
owing  to  the  fact  that  the  peripheral  parts  of  the  object  would  be  more  magnified 
than  the  central  parts.  When  however  a  compound  instrument  is  used  such  as 
that  just  described,  the  objective  forms  a  real  image  showing  "  barrel-shaped 
distortion  "  as  in  fig.  103,  because  the  peripheral  parts  are  less  magnified  than 


FIG.  102  — 
Pincushion  distortion. 


FIG.  103  — 
Barrel-shaped  distortion. 


the  central  parts.  On  now  viewing  this  through  the  second  lens,  or  eyepiece,  the 
barrel- shaped  distortion  will  be  completely  corrected  by  the  tendency  of  the  virtual 
image  to  suffer  from  pincushion  distortion,  so  that  the  final  image  will  be 
rectangular.  The  fact  that  it  is  inverted  is  no  inconvenience. 

The  enormous  advantage  of  a  compound  instrument  is  sufficiently  obvious  from 
this  simple  illustration,  but  it  must  be  remembered  that  the  eyepiece  only  magnifies 


110 


THEORY   OF  THE   MICROSCOPE 


the  detail  that  has  already  been  defined  in  the  real  image  formed  by  the  objective, 
and  that  any  defects  in  this  image  are  exaggerated  by  the  magnification  of  the 
eyepiece. 

2.  Spherical  aberration    Coma. 

As  it  is  of  supreme  importance  to  obtain  the  most  perfect  objective  possible, 
some  of  the  defects  in  the  image  formed  by  a  simple  convex  lens  when  homo- 
geneous light  (light  of  one  specific  wave-length,  i.e.  of  one  colour)  is  used 
will  be  considered  first  and  their  correction  explained,  and  then  the  defects 
in  the  image  due  to  chromatic  aberration  when  ordinary  white  light  is  used 
will  be  very  briefly  referred  to. 

The  defects  in  the  image  formed  by  a  simple  convex  lens  when  homogeneous 
light  is  the  illuminant  are  two  : — spherical  aberration  and  coma. 

Suppose  a  small  bright  point  P  (fig.  104)  to  lie  on  the  axis  of  a  biconvex  lens  ; 
now,  although  the  small  axial  cone  converges  fairly  accurately  to  the  conjugate 
focus  Q,  the  eccentric  rays  PL,  PL'  converge  to  a  nearer  focus  Q'.  The  peripheral 
parts  of  a  lens  refract  light  more  strongly  than  the  central  parts,  and  hence  the 
image  of  the  point  P  will  be  blurred  on  account  of  spherical  aberration.  The  only 
way  of  getting  over  this  difficulty  known  to  the  early  opticians  was  to  cut  off  the 
peripheral  rays  by  means  of  a  diaphragm,  but  this,  of  course,  very  seriously  diminished 
the  brightness  of  the  image. 

Now  take  the  case  where  a  point  P7  (fig.  105)  does  not  lie  on  the  axis  of  the  lens  ; 


FIG.  104. 


FIG.  105. 


the  image  of  P'  will  be  indistinct  for  a  reason  which  is  somewhat  similar  to  that  given 
in  the  former  case,  as  will  be  evident  from  a  glance  at  the  figure.  The  centric  pencil 
will  form  a  well-defined  image  at  Q,  but  while  the  rays  1  and  2  will  intersect  at  A 
the  rays  3  and  4  will  intersect  at  C.  Hence  if  a  screen  be  placed  in  the  position 
QF  a  bright  point  will  be  seen  at  Q,  which  becomes  an  ill- defined  flare 
of  light  towards  F l  (fig.  106).  The  image  somewhat  resembles  the  tail 
of  a  comet  and  the  defect  is  therefore  known  as  coma  (KO/O/,  hair  of 
the  head,  tail  of  a  comet),  and  may  be  regarded  as  the  spherical 
aberration  for  object  points  not  on  the  axis. 

These  two  defects,  spherical  aberration  and  coma,  must  therefore 
be  corrected  before  any  definite  distinct  image  can  be  formed  by 
an  objective.  An  image  free  from  these  defects  is  known  as  an 
aplanatic  image  (<x7r/\ai»js,  not  wandering).  The  condition  for 
FlComa'-~  aplanatism  can  only  be  obtained  in  one  way^-the  lenses  must 
satisfy  what  is  known  as  Abbe's  sine  law. 

The  sine  condition  for  aplanatism. — Let  C  be  the  centre  of  the  circle  KAK'  (fig.  107), 
and  let  P  be  a  point  situated  at  a  distance  CP  from  the  centre  of  the  circle,  such 

that  f-^-rr- 

U-IV  fJt 

.    — 9 

PC     fi' 

i.e.  the  radius  of  the  circle  :  the  distance  of  the  object  to  its  centre 

: :  the  index  of  refraction  of  the  first  medium :  the  index  of  the  second  medium.. 

1  F,  in  this  figure,  is  arbitrary  and  not  the  focus. 

2 In  "primary"  coma  the  angle  formed  by  the  tangents  to  the  series  of  circles  from, 
the  point  Q  in  this  figure  is  said  to  be  60°. 


SPHERICAL   ABERRATION  111 

Now  find  the  point  Q  on  the  axis  such  that 

CK=/?' 

CK     /z     QC 
By  construction  p~r  =  •-,  =7^' 

i.e.  the  sides  of  the  triangles  PCK,  CKQ  about  the  common  angle  C  are  proportional. 


:.  by  Euclid  vi.  6,  the  triangles  are  equiangular,  viz.  CKQ=KPC  and 
KQC=CKP. 

sinCKP    sinCKP     PC     /*'     _ 
shTCKQ  =snTKPC  =CK  =>  =h' 
where  h  is  the  relative  index  of  refraction  of  the  second  medium  to  the  first  medium. 

But  if  P  be  a  source  of  light, 

sin  CKP  =sin  <£  =h  sin  $'  —h  sin  CKQ, 
where  <£=  angle  of  incidence,  and  </>'  angle  of  emergence. 

.'.   KR  must  be  the  refracted  emergent  ray. 

Now  the  position  of  K  is  arbitrary  ;  therefore  all  the  light  diverging  from  P, 
however  great  the  angle  U,  must  after  refraction  appear  to  come  from  Q.  (The 
angle  U,  or  CPK,  represents  of  course  merely  one-half  the  cone  of  light  that  falls 
on  the  lens.) 

This  then  satisfies  the  first  condition  of  aplanatism  ;  in  other  words,  the  spherical 
aberration  is  corrected. 

With  an  oil-immersion  lens,  seeing  that  the  oil  and  the  glass  have  practically  the 
same  index  of  refraction,  the  object  may  for  all  practical  purposes  be  regarded  as 
in  the  glass,  which  is  exactly  the  condition  required  to  satisfy  the  sine  condition. 

For  the  second  condition,  —  the  correction  of  coma: 

From  centre  C  at  distances  CP  and  CQ  draw  arcs  PB  and  QB'  (fig.  107).  Join 
CBB'.  Then  CBB'  may  be  regarded  as  the  axis  of  the  lens,  and  the  image  of  B  will 
be  formed  at  B'. 

Regard  PB  as  the  object  o,  and  QB'  as  the  image  i. 


i     QB'    QC    //CK 
o~PB~PC~  PC    "//sinCKP 
p.  sin  KPC      fi  sin  U 


~  fj.'  sin  KQC  ~  jtx'  sin  U'  ' 
that  is, 

index  of  refraction  of  first  medium  x  sin  of  hah*  the 

dimensions  of  the  image  _  _  angle  of  the  rays  diverging  from  the  object 
dimensions  of  the  object  ~  index  of  refraction  of  final  medium  x  sin  of  hah*  the  " 

angle  of  convergence  of  the  rays  forming  the  image 
and  this  is  the  one  and  necessary  test  for  aplanatism. 


112  THEORY   OF  THE  MICROSCOPE 

3.  Angular  aperture. 

The  angle  U  (fig.  107)  is  the  semi-aperture  of  the  lens;  and  the  total 
aperture  (2U)  is  the  angle  formed  by  the  two  extreme  rays,  which  starting 
from  the  same  point  on  the  object  ultimately  reach  the  eye  of  the  observer. 
And  obviously,  the  greater  the  angle  of  aperture  the  greater  will  be  the 
number  of  rays  of  light  which  leaving  the  same  point  on  the  object  reach 
the  eye  of  the  observer,  and  consequently  the  brighter  will  be  the  image  of 
a  given  size.  So  that  it  is  important,  especially  with  the  more  highly  magni- 
fying lenses,  that  the  angle  of  aperture  should  be  as  large  as  possible.  Lenses 
are  now  made  whose  angular  aperture  is  140°  or  even  more.  The  angle 
of  aperture  is  measured  with  the  aid  of  a  special  piece  of  apparatus  known 
as  an  apertometer. 

4.  Numerical  aperture. 

The  expression  /A  sin  U  is  commonly  known  as  the  numerical  aperture  of  the 
lens,  and  is  denoted  by  N.A. 

It  will  be  easily  seen  that  the  brightness  of  the  image 
varies  amongst  other  things  as  (N.  A.  )  2.  For  the  numerical 
aperture  determines  the  amount  of  light  entering  in  one 
diametral  plane  of  the  objective,  and  therefore  the  total 
amount  of  light  entering  the  circular  objective  must  vary 
as  (N.A.)2. 

Lenses  are  more    commonly  described    by  their   nu- 


merical aperture  than  by  their  angular  aperture.  The 
N.A.  of  a  ^-in.  dry  lens  should  not  be  less  than  *82,  and 
of  a  TV-in.  oil-immersion  lens  not  less  than  1*3. 

The  N.A.  is  determined  thus :    Suppose  a  dry  objective  has  an  angular  aperture 
of  60°. 

Let  U=^  angular  aperture,  and  since  the  refractive  index  of  air  is  1, 

N.A.=^sinU 
=  1  sin  30° 
=  •5. 

Again,  suppose  an  oil-immersion  objective  has  a  total  angular  aperture  of  135°. 
U=i(135°)=67£°    and    sin  67^°  =  "9238795. 

H  for  cedar- wood  oil  =  1  *52 ; 
then  N.A.  =IJL  sin  U, 


^Tl'404; 

and  if  the  same  lens  be  used  dry,  since  the  critical  angle  for  glass  is  41°,  the  total 
effective  angular  aperture  would  be  82°. 

H  for  air  =  1,    and   sin  41°  =  '656059  ; 
/.    N.A~'656. 

5.  Resolving  power. 

The  resolving  power  of  a  lens  is  the  capacity  of  the  lens  to  optically  separate 
two  closely  adjacent  points  on  an  image  which  the  unaided  eye  is  unable  to 
distinguish  as  separate,  and  must  be  carefully  distinguished  from  magnifying 
power. 

It  is  found  1  that  two  objects  at  a  distance  d  apart  can  be  separated  by  oblique 
illumination  if 

•61  A          -61  A 
~2/z  sinU~2N.A.; 

1  TkelTheory  of  Optical  Instruments,  by  E.  T.  Whittaker,  M.A.,  F.R.S.  ;  Camb.  Univ. 
Press  ;  2s.  6d. 


BRIGHTNESS   OF  IMAGE 

and  by  direct  illumination  if 


113 


d  = 


•61 A 
N.A. 


where  A  =wave  length  of  light  used. 
Thus,  in  the  middle  of  the  spectrum, 

A  =  -00054  mm., 
and  N.A.  in  the  very  best  lenses  =1*6  ; 


from  which  it  is  apparent  that  it  is  impossible  to  distinguish,  i.e.  to  resolve,  any 
two  points  less  than  '000103  mm.  (approximately  0'1/x)  apart,  or  to  see  the  details 
of  an  object  of  smaller  dimensions  than  O'l/u. 

Limit  of  effective  magnification.  —  Now  the  eye  can  only  easily  distinguish 
two  objects  as  separate  whose  distance  apart  subtends  an  angle  of  2'  at  its 
nodal  point.  This  is  the  angle  which  a  distance  of  '1477  mm.  subtends  at 


10  inches.    Then  the  necessary  magnification  is  : 


•1477 
0001 ' 


1477.      Consequently, 


the  limit  of  resolution  of  the  microscope  is  attained  when  the  total  magnifica- 
tion is  about  1450.  With  an  high  eyepiece  a  further  magnification  may  be 
obtained  up  to  1600  or  2000,  or  even  3000,  but  no  more  detail  will  be  dis- 
coverable. The  effect  of  the  higher  magnification  will  merely  make  the 
detail  larger :  it  will  add  no  new  detail  but  will  still  further  diminish  the 
brightness  of  the  image. 

Resolving  power  oc    ,  for  it  varies  inversely  as  the  least  distance  between 

Cv 

separable  points.     Hence,  whatever  the  focus,  the  resolving  power  oc  N.A.,  and 

(N  A  )2 
comparing  lenses  of  the  same  focus,  the  brightness  of  the  image  oc  v    '        • 


6.  Brightness  of  image. 

Suppose  M  and  M'  be  the  magnifications  obtained,  using  the  same  objective 
but  different  eyepieces, 
and  let  M'  =2500,  and  M  =  1450. 

T>/ 

The  relative  brightness  -^  of  the  image  in  the  two  cases  will  be 
B 

IT     (N.A)MN.A.)2     (1450)2  _1 

B*     M'2  M2     °c(2500j2'  ~"3; 

so  that  the  penalty  of  increasing  the  magnification  from  1450  to  2500  is  to  make 
the  brightness  of  the  image  -J  what  it  was  with  the  lower  magnification. 

The  penetrating  power  oc  =^-r-,  so  that 
N.A. 


Resolving  power 
a  N.A. 

Brightness  of  image 
oc(N.A.)- 

Penetrating  power 

«j& 

(Oil 
Ratio 
iDry 

1-404 
•656 

1-972 
•43041 

•7121 
1-5242 

(Oil 
or 
I  Dry 

2-14 
1 

4-58 

1 

1 
2-14 

114  THEORY   OF   THE   MICROSCOPE 

That  is  to  say  the  resolving  power — the  capacity  to  recognize  as  distinct 
two  closely  adjacent  points — is  more  than  twice  as  great,  and  the  brightness 
of  the  image  more  than  4J  times  as  great  when  the  same  lens  is  -used  with 
oil  as  when  used  dry  ;  while  as  regards  penetrating  power  a  dry  lens  is  more 
than  twice  as  efficient  as  an  oil  lens,  so  that  when  thick  sections  have  to  be 
examined  a  dry  objective  of  low  N.A.  should  be  selected. 

And  it  is  clear  that  the  N.A.  is  of  fundamental  importance  in  determining 
the  efficiency  of  a  microscope. 

7.  Definition. 

The  definition  of  a  lens  is  its  capacity  to  render  the  outline  of  an  object 
or  image  distinct  to  the  eye,  and  depends  partly  upon  the  sufficiency  of  the 
correction  for  aplanatism,  which  can  be  assisted  by  the  use  of  diaphragms, 
and  partly  upon  the  sufficiency  of  the  correction  for  chromatic  aberration  : 
from  which  it  follows  that  an  achromatic  lens  has  a  better  definition  than  a 
lens  not  so  corrected. 

The  definition  and  resolving  power  of  a  lens  are  in  practice  tested  by  means  of 
preparations  of  diatoms,  those  generally  used  for  the  purpose  being  Pleurosigma 
angulatum,  Grammatophora  subtilissima,  Navicula  crassinervis,  Surdrella  gemina, 
etc.  With  a  good  objective  a  very  distinct  image  with  sharply  defined  outlines  will  be 
obtained ;  in  the  case  of  Pleurosigma  angulatum  it  should  be  possible  to  make  out, 
under  a  magnification  of  500-600  diameters,  a  central  venule  on  to  which  two 
systems  of  oblique  lines  abut,  crossing  each  other  at  an  acute  angle  and  forming  a 
reticulated  system  of  fine  lines. 

It  is  also  well  when  testing  an  objective,  to  examine  some  small  organism  such 
as  the  Bacillus  tuberculosis,  in  order  to  ascertain  the  magnification  produced  as 
well  as  the  sharpness  of  the  image. 

8.  Chromatic  aberration— achromatism  and  apochromatism. 

So  far  the  conditions  which  must  be  fulfilled  by  a  lens  when  homogeneous 
light  is  the  ilmminant  have  been  considered.  But  in  practice  white  or  non- 
homogeneous  light,  i.e.  light  of  different  wave  lengths,  is  used.  And  with 
white  light  a  series  of  images  will  be  formed  of  different  colours,  in  different 
places,  and  of  different  sizes.  Further,  only  one  of  these  images  corresponding 
to  one  definite  wave  length  will  be  aplanatic.  As  will  be  readily  appreciated, 
the  calculations  required  for  the  correction  of  chromatic  errors  are  of  necessity 
extraordinarily  complex  ;  it  must  therefore  suffice  here  to  say  that  in  practice 
chromatic  aberration  is  corrected  by  the  use  of  lenses  combined  in  pairs  (or 
triplets),  one  lens  being  concave  the  other  convex.  The  convergent  convex 
lens  is  made  of  crown  glass,  which  has  a  low  dispersive  power,  while  the 
divergent  concave  lens  is  made  of  flint  glass,  which  has  a  high  dispersive 
power.  By  making  these  two  lenses  of  a  suitable  curvature  the  chromatic 
aberration  for  two  colours  is  corrected,  and  the  lenses  are  said  to  be  achro- 
matized for  those  colours. 

If  57  denote  the  dispersive  power  of  the  glass  between  the  F  and  C  lines  of  the 
spectrum,  and  /  denote  the  focal  length  of  the  lens,  then,  in  order  to  achromatize 
the  blue  and  red  colours  F  and  C,  the  couplet  must  be  such  that  £T2/i—  ~~^i/2' 

In  apochromatic  (UTTO,  apart  from;  x/°<Va>  colour)  couplets,  fluorite  takes  the 
place  of  crown  glass.  Fluorite  has  a  similar  relative  dispersion  to  flint,  so 
that  with  these  couplets  3  (not  2)  different  colours  can  be  achromatized. 
Suppose  that  in  a  given  apochromatic  system  the  focal  lengths  for  the  red, 
the  yellow,  and  the  green  rays  are  the  same  :  then  the  magnifications  will  be 
the  same,  but  the  images  will  not  all  lie  in  one  plane.  Again,  suppose  a 
system  were  so  constructed  that  all  the  different  colours  should  come  to  a 


CHROMATIC   ABERRATION 


115 


focus  in  the  same  plane  :  then  the  images  would,  though  superposed,  be  of 
different  sizes.  And  in  order  to  correct  as  far  as  possible  these  defects,  an 
under  correction  in  one  couplet  is  compensated  by  an  over-correction  in  the 
next.  Abbe's  apochromatic  oil-immersion  objective  is  made  up  of  ten  lenses, 
as  illustrated  in  the  figure.  Even  then  the  achromatism  is  only  carried  out 
with  regard  to  the  position  of  the  image,  not  to  its  size.  Without  entering 


FIG.  109.  —  Abbe's  apochromatic  oil-immersion  objective. 

upon  a  discussion  of  the  calculations  necessary  to  determine  the  curvatures 
of  the  different  lenses,  it  may  be  said  that  in  practice  if  an  objective  be 
over-corrected,  that  is  if  the  power  of  the  flint  glass  be  too  great  in  pro- 
portion to  that  of  the  crown  glass,  the  error  may  be  rectified  by  slightly 
separating  the  lenses  by  means  of  a  compensating  collar  :  this  has  practi- 
cally the  effect  of  decreasing  the  power  of  the  flint  lens.  Now  if  an  object 
be  examined  under  a  microscope  with  an  achromatic  objective  it  will  be 
found  that  the  image  has  either  a  bluish  outline  or  a  yellowish  outline. 
The  former  is  the  more  common  defect,  and  is  due  to  over-correction,  while 
the  latter  is  a  result  of  under-correction.  Abbe's  apochromatic  objective 
being  achromatic  for  three  colours,  is  free  from  secondary  spectra  ;  since 
however  the  achromatism  has  regard  to  the  position  of  the  images,  not 
to  their  sizes,  the  blue  image  though  formed  in  the  same  plane  as  the  red 
image  is  larger  than  the  latter.  This  error  is  subsequently  corrected  by  the 
compensating  ocular,  which  produces  larger  red  images  than  blue.  More- 
over the  sine  condition  is  attained  for  two  colours  so  that  each  of  these 
images  is  aplanatic,  i.e.  the  image  formed  by  these  two  colours  is  free  from 
spherical  aberration  and  coma.  It  will  therefore  be  seen  that  no  lens  is, 
in  the  strict  sense  of  the  word,  achromatic. 

Apochromatic  lenses  with  the  necessary  compensating  eyepieces  are  very 
expensive,  and  are  only  necessary  for  special  work.  Ordinary  achromatic 
objectives  are  quite  sufficient  for  general  purposes. 

9.  Flatness  of  image. 

Even  now  all  the  defects  of  the  image  formed  by  a  simple  lens  have  not 
been  studied,  for  it  will  always  be  found  that  the  image  is  curved.  To  secure 
flatness.  of  the  image,  a  condition  known  as  Petzval's  condition  must  be 
satisfied. 

Petzval's  condition  for  flatness  of  image.  —  A  couplet  must  satisfy  the  condition 
that 


But  the  essential  condition  for  achromatism  is 


116  THEORY   OF  THE   MICROSCOPE 

It  is  necessary  therefore  to  use  glass  with  a  high  refractive  index  but 
low  dispersive  power  to  obtain  an  achromatic  flat  image.  It  is  only  recently 
that  Messrs.  Schott  of  Jena  have  succeeded  in  making  such  a  glass — barium 
silicate  glass — which  produces  a  greater  refraction  and  a  smaller  dispersion 
than  crown  glass. 

B.  The  eyepieces. 

An  eyepiece  is  a  system  of  lenses  so  arranged  that  the  real  image  produced 
by  the  objective  in  the  tube  is  magnified  and  trans- 
mitted to  the  eye  of  the  observer. 

In  practice  it  is  found  expedient  to  form  the  eyepiece 
of  two  plano-convex  lenses  separated  by  an  interval ; 
the  lower  lens  is  called  the  field-lens,  for  it  increases 
the  field  of  view  of  the  instrument,  the  upper  lens 
is  called  the  eye-lens. 

The  eyepiece  most  commonly  used  is  that  known 

FIG.  110.— Theory  of  Huy-  ,,      __-7  .  .  J 

genian  eyepiece.  as  the  Huygenian  eyepiece. 

(To  render  the  figure  less        In  its  simplest  form  this  eyepiece  consists  of  two 

otS?\vi&e\ppear,  theprin-  plano-convex  lenses — a  field-lens  F,    and  an  eye-lens 

cipal  plane  is  taken  as  on  E      Tne  focai  iength  of  the  former  /,  is  three  times 

the  flat  side  of  the  lens.)  0,11,          Prm  a          J  r         11 

that  of  the  latter /2.     The  curved  surface  of  each  lens 

faces  the  incident  light,  and  the  lenses  are  separated  by  an  interval  d  which 
is  twice  the  focal  length  of  the  eye-lens. 

Thus  fl  =3/,,    and   d  =  -  2/,. 

Let  ba  (fig.  110)  represent  the  aplanatic  image  of  the  object  formed  by  the  objec- 
tive. The  field- lens  of  the  eyepiece  is  placed  below  it,  indeed  half  its  focal  length 
below  it.  Consequently  the  image  ba  is  not  actually  formed,  but  the  converging 
pencils  proceeding  towards  the  separate  points  of  the  image  ba  are  made  by  the 
field-lens  to  converge  towards  the  separate  points  of  the  image  b'a'.  Now  since 
the  cone  of  light  that  corresponds  to  any  point  of  the  image  ba  meets  only  an 
exceedingly  small  portion  of  the  field-lens,  we  may  neglect  the  aberrations  which 
occur  within  each  of  these  cones.  Therefore  we  may  regard  each  point  of  the 
image  b'a'  as  being  fairly  distinct ;  but  it  is  necessary  to  consider  in  what  way 
the  spherical  aberration  of  the  field-lens  will  affect  their  relative  positions.  Now  the 
field-lens  may  be  regarded  as  consisting  of  several  annular  zones,  the  refracting  power 
of  each  zone  increasing  with  its  distance  from  the  centre.  The  axial  ray  of  the 
peripheral  pencil  aa'  will  consequently  undergo  a  greater  deviation  than  that  of  the 
intermediate  pencil  such  as  yc.  The  consequence  of  this  will  be  that  the  image 
b'a'  will  be— 

1.  Curved,  because  the  refracting  power  of  the  peripheral  portion  of  the  field- lens 
being  greater  than  the  more  central  portion,  the  focus  a'  of  the  peripheral  pencil 
will  be  nearer  the  lens  than  the  focus  c  of  the  intermediate  pencil. 

2.  Distorted,  for  the  peripheral  parts  of  the  image  ca'  will  be  smaller  than  the 
more  central  part  b'c,  i.e.  the  distortion  is  barrel-shaped  (p.  109). 

3.  Smaller  than  the  image  ba. 

In  the  majority  of  text- books  the  Huygenian  eyepiece  is  also  proved  to  be 
achromatic.  But  the  proof  is  only  applicable  for  incident  parallel  rays.  The  eye- 
piece does  not  give  a  strictly  achromatic  image  of  the  objective's  image,  so  that 
the  proof  is  not  worth  considering.  It  has  already  been  said  that  the  apochromatic 
objective  forms  larger  blue  images  than  red.  The  compensating  eyepiece  having 
the  eye-lens  of  a  flint  and  crown  glass  combination  forms  larger  red  images  than 
blue,  and  consequently  the  final  image  is  completely  achromatic. 

It  will  be  obvious  from  what  has  been  said  that  there  are  so  many  errors 
to  correct  that  it  would  appear  well  nigh  impossible  to  correct  them  all.  So 
would  it  be  were  it  necessary  to  form  a  point  image  on  the  retina  of  each 
point  of  the  object ;  fortunately  the  structure  of  the  retina  obviates  this 
necessity.  The  smallest  visual  area  of  the  retina  is  a  retinal  cone.  In  the 


THE   CARE   OF   THE   MICROSCOPE  117 

region  of  most  distinct  vision,  the  fovea,  the  cross  section  of  a  cone  is  a  cir- 
cular area  of  diameter  '002  mm.  Now  if  not  more  than  one  cone  is  stimulated 
the  resulting  impression  will  be  that  of  a  single  point  of  light.1  Therefore 
errors  will  be  absolutely  negligible  if  they  give  rise  to  such  small  confusion 
circles  that  they  do  not  extend  over  more  than  one  foveal  cone. 


SECTION  III.— THE   CARE   OF   THE   MICROSCOPE. 

The  microscope  must  be  kept  at  as  uniform  a  temperature  as  possible  and 
away  from  direct  sunlight  and  all  other  sources  of  heat,  because  the  lenses 
are  held  in  position  by  Canada  balsam,  and  they  would  be  displaced  and  the 
instrument  put  out  of  order  if  the  balsam  were  to  be  melted.  It  is  also 
essential  to  protect  the  microscope  from  dust,  which  may  best  be  done  by 
standing  it  on  a  piece  of  thick  felt  or  india-rubber  on  the  bench,  and  cover- 
ing it  when  not  in  use  with  a  glass  shade. 

Objectives  and  eyepieces  should  always  be  wiped  with  a  piece  of  soft  linen 
before  use,  to  ensure  their  being  absolutely  clean.  If  on  looking  down  the 
microscope  a  speck  of  dust  be  seen  in  the  field,  one  must  find  out  where  it 
is  in  order  to  wipe  it  off.  To  determine  the  position  of  the  speck,  first  rotate 
the  eyepiece ;  if  the  dust  be  on  one  or  other  of  these  lenses  it  will  of  course 
alter  its  position  ;  and  if  rotation  of  the  eyepiece  do  not  alter  its  position, 
then  it  is  on  the  objective.  By  holding  the  lenses  up  to  the  light  some 
distance  from  the  eye,  it  can  be  seen  if  they  are  cloudy  or  if  specks  of  dust 
adhere  to  them. 

To  clean  the  front  lens  of  the  objective,  rub  it  with  an  absolutely  clean 
piece  of  fine  linen  ;  if  this  fail  to  clean  it,  take  a  piece  of  elder  pith,  strip  off 
a  thin  layer,  and  with  the  clean  surface  so  exposed  gently  rub  the  lens. 

If  cedar-wood  oil,  Canada  balsam,  or  dammar  varnish  be  sticking  to  the 
lens,  moisten  the  cloth  with  a  drop  of  xylol,  and  gently  wipe  the  lens.  An 
excess  of  xylol  must  not  be  used  nor  should  xylol  be  poured  on  to  the  objec- 
tive, for  fear  that  it  should  penetrate  between  the  lenses  and  their  mountings 
and  dissolve  the  balsam  holding  them  in  position. 

When  it  is  necessary  to  examine  preparations  in  caustic  potash,  acids  or 
other  chemical  reagent,  great  care  must  be  taken  to  keep  the  lenses  from 
coming  in  contact  with  the  reagent :  but  if  by  accident  the  lens  should  be 
soiled,  wash  it  at  once  in  distilled  water  and  dry  with  a  soft  linen  rag. 

If  the  objective  be  cloudy,  and  cleaning  the  outer  lens  does  not  remove 
the  cloudiness,  it  must  not  be  unscrewed  to  clean  the  inner  lens,  but  should 
be  sent  to  the  maker,  who  is  the  only  person  capable  of  putting  it  right. 

Objectives  should  be  carefully  protected  against  the  slightest  shocks 
or  falls. 

The  eyepiece  and  the  Abbe  condenser  can  be  cleaned  in  the  same  way  as 
the  objective,  but  these  lenses  are  much  more  accessible  and  infinitely  less 
delicate.  The  mirror  can  also  be  cleaned  in  the  same  way. 

Before  putting  the  microscope  away,  always  wipe  the  eyepiece  and  objec- 
tives, and  remove  every  trace  of  oil  from  the  immersion  lens. 

The  stand  should  be  wiped  frequently  with  a  chamois  leather,  and  rubbed 
in  the  direction  in  which  the  lacquer  has  been  applied.  Should  the  stand 
be  accidentally  soiled  with  balsam  or  cedar-wood  oil,  apply  a  little  xylol  on 
a  soft  cloth,  and  remove  it  at  once  with  a  chamois  leather  ;  if  too  much  xylol 
be  used  or  if  it  be  not  carefully  wiped  off  it  will  dissolve  the  lacquer  from 
the  metal. 

1  For  simplicity  of  explanation  the  question  of  diffraction  is  ignored  in  this  case. 


118  PRACTICAL  DIRECTIONS 

A  little  xylol  can  also  be  used  to  clean  the  stage. 

The  coarse  and  fine  adjustments  should  be  lubricated  from  time  to  time  by 
the  application  of  a  trace  of  vaseline. 

SECTION  IV.— METHOD   OF  USING  THE  MICROSCOPE. 
1.  The  source  of  light. 

The  microscope  when  in  use  should  rest  on  a  firm  table  in  front  of  a  window. 
The  best  light  for  microscope  work  is  that  reflected  from  a  white  cloud,  but 
the  light  may  be  taken  also  from  a  clear  sky  or  a  white  wall.  Direct  sunlight 
is  totally  unsuitable. 

In  default  of  a  satisfactory  natural  light,  a  good  petrol-air  or  albo-carbon 
lamp  may  be  used,  though  a  lamp  such  as  Ranvier's  with  an  Auer  burner  is 
better.  With  these  lamps  it  is  sometimes  necessary  to  interpose  a  sheet  of 
ground  glass  between  the  source  of  light  and  the  microscope  to  moderate  the 
intensity  of  the  former. 

[Many  observers  prefer  a  small  oil  lamp,  but  for  general  use  a  very  satis- 
factory artificial  light  is  to  be  obtained  by  the  use  of  an  inverted  incandescent 
gas  mantle,  the  light  from  which  is  passed  through  a  large  flask  filled  with 
distilled  water  before  reaching  the  mirror  (fig.  111).] 


FlQ.  111. — Illumination  with  an  inverted  incandescent  gas  burner. 

Turn  the  microscope  towards  the  source  of  light,  look  down  the  tube,  and 
taking  hold  of  the  sides  of  the  mirror  move  the  latter  about  until  the  field 
is  brightly  illuminated. 

1.  With  dry  lenses  use  a  concave  mirror,  which  throws  a  convergent  pencil 
of  light  on  to  the  object. 

2.  When  using  an  immersion  lens,  it  is  necessary  to  have  an  Abbe  condenser 
fitted  below  the  stage.     With  a  condenser  aflat  mirror  must  always  be  employed  ; 
the  rays  reflected  from  the  flat  mirror  are  converged  by  the  condenser  and 
brought  to  a  focus  on  the  object,  so  that  by  the  use  of  a  condenser  a  con- 
siderable amount  of  light  is  obtained. 

Every  microscope  should  be  provided  with  a  diaphragm  below  the  stage. 
The  size  of  the  opening  in  the  diaphragm  will  be  determined  by  the  magnifi- 
cation employed ;  the  greater  the  magnification  the  smaller  should  be  the 
opening  in  the  diaphragm.  By  cutting  off  the  marginal  rays — which  are 
not  only  useless  but  actually  detract  from  the  sharpness  of  the  image — the 
diaphragm  assists  in  the  correction  of  spherical  aberration,  and  produces  a 
sharper  definition  of  the  object. 


IMMERSION   LENSES  119 

2.  Arrangement  of  the  object. 

The  object  to  be  examined  under  the  microscope  must  be  mounted  on  a 
microscope  slide — a  thin  very  transparent  piece  of  glass  free  from  bubbles 
of  air — and  may  be  covered  with  a  cover-glass— a  much  thinner  and  smaller 
piece  of  glass,  square  or  circular  in  shape,  and  measuring  18-25  mm.  in 
diameter,  but  not  exceeding  0' 15-0' 20  mm.  in  thickness. 

The  rays  of  light  coming  from  the  object  as  they  pass  through  the  cover-glass 
will  be  displaced  to  a  greater  or  less  extent,  depending  upon  the  thickness  of  the 
glass.  Fig.  112  shows  this.  Given  any  point  A  on  the  object,  its  image  on  account 
of  displacement  will  appear  along  the  line  DE,  and 
will  be  diffuse ;  with  [dry]  high-power  lenses  especi- 
ally, much  of  the  brightness  and  sharpness  of  the 
image  will  be  lost. 

[To  obtain  perfect  definition  with  the  higher 
powers  of  the  microscope,  the  thickness  of  the 
cover-glass  is  important,  and  for  two  reasons  : 

1.  "If  the  cover-glass  be  very  thick,  there  may 
not    be    room    enough    to    bring    the   front    lens 
sufficiently  near  to  focus  the  specimen. 

2.  "  The  varying  thickness  of  the   actual  glass 
introduces  errors  in  the  adjustment  of  the  com- 
ponents  of  the  lens  system  »  (Spitta).] 

To  overcome  this  difficulty,  it  is  only  necessary   glass. 
to  use  cover-glasses  of  the  thickness  indicated  on 

the  objective,  each  objective  being  corrected  to  work  for  a  given  thickness.  Or, 
since  cover-glasses  of  exactly  the  same  thickness  cannot  always  be  obtained,  one 
may  have  objectives  of  certain  magnifications,  which  can  be  corrected  by  altering 
the  distance  between  the  component  lenses :  the  thicker  the  cover-glass  the  nearer 
must  the  lenses  be  together. 

But  now  that  all  microscopes  have  a  draw  tube  this  correction  is  really  not  of 
vital  importance,  because  by  altering  the  length  of  the  tube  the  effect  of  the  thick- 
ness of  the  cover-glass  can  within  certain  limits  be  counteracted.  The  thicker  the 
cover-glass  the  shorter  must  the  tube  be.  With  the  draw  tube  right  down  in  its 
socket,  cover-glasses  0'25  mm.  thick  can  be  used,  but  with  a  normal  length  of  tube 
(160-170  mm.)  one  must  have  cover-glasses  no  thicker  than  0'15  to  0'18  mm. 

3.  Homogeneous  immersion  lenses. 

Immersion  lenses  are  used  in  order  to  counteract  the  refraction  of  rays 
of  light  in  passing  from  glass  to  air.  In  using  an  immersion  lens  a  drop  of 
some  liquid,  the  refractive  index  of  which  is  as  nearly  as  possible  the  same 
as  that  of  glass,  is  placed  on  the  cover-glass,  and  the  lens  lowered  into  it. 
Cedar- wood  oil  has  a  refractive  index  of  T515  to  1'520,  a  mixture  of  castor- 
oil  and  essence  of  anise  about  1-510,  and  monobromonaphthaline  T66. 
Homogeneous  immersion  objectives  do  not  need  to  be  corrected. 

When  rays  of  light  pass  from  the  cover-glass  into  air,  their  direction  is  altered 
in  such  a  manner  that  all  rays  making  with  the  surface  of  the  cover-glass  a  smaller 
angle  than  [48°  12']  are  totally  reflected  and  are  lost  to  the  objective.  By  sub- 
stituting a  substance  of  the  same  refractive  index  as  glass  for  air,  this  loss  of  light 
is  avoided.  An  immersion  lens  makes  the  image  very  much  [brighter  and] 
sharper ;  so  that  an  homogeneous  immersion  objective  whose  angle  of  aperture 
measures  82°  has  the  same  value  (i.e  numerical  aperture)  as  a  dry  lens  whose 
angle  of  aperture  is  180°  (//,  sinU)  (p.  112).  Moreover  for  the  same  magnification 
an  immersion  objective  has  a  greater  focal  length  than  a  dry  lens. 

It  is  necessary  to  use  an  Abbe  condenser  with  an  immersion  lens,  and 
perfect  results  can  only  be  obtained  with  a  given  length  of  tube  (generally 
160-170  mm,).  A  drop  of  cedar-wood  oil  is  placed  on  the  cover-glass,  and  the 
objective  is  lowered  until  its  front  lens  touches  the  oil. 


120  PRACTICAL   DIRECTIONS 

Immersion  lenses  should  be  used  only  with  stained  preparations.  They  are 
not  suitable  for  the  examination  of  unstained  preparations,  because  the 
light  focussed  by  the  condenser  is  so  intense  that  it  drowns  unstained  objects 
and  renders  their  outline  very  indistinct. 

4.  The  nosepiece. 

The  nosepiece  in  most  general  use  is  constructed  to  carry  three  objectives 
— usually  a  No.  2,  and  a  No.  8  or  No.  9  dry,  and  a  T\vth  homogeneous  immer- 
sion lenses.  Each  objective  is  screwed  into  its  proper  place  in  the  nosepiece, 
which  is  marked  for  the  purpose ;  it  is  necessary  that  this  be  done  in  order 
to  get  the  centering  true.  By  simply  rotating  the  nosepiece,  it  is  thus  possible 
without  unscrewing  them  to  use  any  of  the  objectives. 

5.  Eyepieces. 

In  the  great  majority  of  cases  a  low-power  eyepiece  should  be  used.  A 
high-power  eyepiece  only  magnifies  at  the  expense  of  brightness  and  sharp- 
ness (p.  113).  Eyepieces  I.  or  II.  are  generally  used,  III.  and  IV.  only  when 
delicate  work  requiring  considerable  magnification  is  in  hand. 

6.  Focussing. 

Focussing  is  done  in  two  stages.  The  object  is  first  brought  approximately 
into  focus  with  the  coarse  adjustment,  and  then  sharply  focussed  by  means 
of  the  fine  adjustment. 

The  focal  length  varies  with  the  different  objectives,  being  in  inverse  ratio 
to  the  magnification.  The  approximate  focus  for  each  objective  is  soon 
learnt  with  a  little  practice,  so  that  the  first  stage  of  the  process  is  quickly 
done. 

The  object  having  been  brought  more  or  less  into  focus  with  the  aid  of 
the  coarse  adjustment,  is  exactly  focussed  by  means  of  the  fine  adjustment 
working  on  a  micrometer  screw. 

When  high  powers  are  used  the  objective  will  be  close  to  the  cover-glass, 
and  a  rough  movement  downwards  of  the  lens  will  most  certainly  break  the 
slide.  [Microscopes  are  now  made  so  that  it  is  impossible  to  force  the  objec- 
tives through  the  cover-glasses.]  In  any  case,  to  avoid  this  possibility 
proceed  as  follows : 

1.  Before  looking  down  the  microscope,  fix  the  eye  on  the  preparation, 
and  lower  the  tube  slowly  with  the  coarse  adjustment  until  the  front  lens 
touches  the  cover-glass. 

2.  Now  look  down  the  tube,  and  raise  the  coarse  adjustment  until  the 
preparation  is  approximately  focussed. 

3.  Then  get  the  exact  focus  by  gently  rotating  the  fine  adjustment. 

The  fine  adjustment  should  never  be  used  for  large  alterations  of  focus ;  it  is  a 
very  sensitive  and  delicate  screw,  acting  on  the  microscope  tube  through  a  spiral 
spring,  which  would  soon  be  put  out  of  gear  if  used  for  large  excursions. 

While  the  fine  adjustment  is  being  used,  the  thumb  and  index  finger  of  the 
right  hand  should  not  be  taken  off  the  micrometer  screw,  but  should  con- 
tinually move  it  backwards  and  forwards  gently,  until,  without  any  effort 
of  accommodation,  the  different  parts  of  the  preparation  are  brought  into 
focus  and  seen  in  succession,  and  the  shape  of  the  object  distinctly  made  out. 

While  examining  a  preparation,  the  slide  should  be  held  between  the 
thumb  and  index  finger  of  the  left  hand,  and  moved  about  on  the  stage,  so 
that  the  different  parts  can  be  brought  within  the  field  as  required.  [It  is  a 
great  advantage  to  have  a  "  mechanical  stage  "  fitted  to  the  microscope ; 


MEASUREMENT   OF   OBJECTS  121 

this  enables  the  observer  by  turning  a  milled  screw  to  place  with  ease  and 
accuracy  any  portion  of  the  slide  under  the  objective,  and  by  continuous 
rotation,  rapidly  to  examine  the  whole  preparation.] 


SECTION  V.—  THE    MEASUREMENT    OF    MICROSCOPICAL    OBJECTS. 

1.  The  experimental  determination  of  the  magnification 
produced  by  a  system  of  lenses. 

Magnification  produced  by  an  optical  system  is  of  course  magnification 
in  diameters. 

Microscope  makers  supply  a  table  with  each  of  their  instruments,  showing 
with  a  given  tube  length  the  magnification  produced  with  every  combination 
of  objective  and  ocular.  This  table  may  be  verified  roughly  by  one  or  other 
of  the  two  following  methods  : 

A.  With  a  camera  lucida.  —  For  this  purpose  a  camera  lucida  and  a  stage 
micrometer  are  necessary.  A  stage  micrometer  is  a  thin  glass  slide,  on 
which  a  scale  mechanically  divided  by  parallel  lines  into  TJ^ths  of  a 
millimetre  has  been  engraved. 

1.  Select  the  eyepiece  and  the  objective  of  which  the  magnification  produced 
by  the  combination  is  to  be  determined.     Lengthen  the  tube  to  160  mm., 
or  whatever  is  the  proper  working  length.     Place  the  micrometer  slide  on 
the  stage  and  get  it  into  focus,  so  that  the  divisions  on  the  scale  are  sharply 
defined. 

2.  Place  a  sheet  of  paper,  bluish  for  choice,  on  a  small  drawing  table  level 
with  the  stage  of  the  microscope  on  the  right-hand  side  of  the  instrument. 
Fit  the  camera  lucida  to  the  eyepiece. 

3.  On  looking  down  the  tube  of  the  microscope,  two  images  of  the  scale 
on  the  micrometer  will  be  seen  —  one  formed  directly  by  rays  passing  through 
the  camera  lucida,  the  other  projected  by  reflection  at  the  prism  on  to  the 
paper.     If  the  image  projected  on  to  the  paper  be  approached  with  the  point 
of  a  pencil,  the  latter  will  also  come  into  view,  and  it  will  be  easy  to  outline 
on  the  paper  the  position  of  the  image  of  the  scale  on  the  micrometer.     Trace 
the  position  of  a  few  of  the  divisions  of  the  scale. 

4.  With  a  millimetre  scale  measure  the  distance  between  any  two  of  the 
lines  sketched. 

Let  n  =the  distance  in  millimetres  of  two  adjacent  divisions,  and  M  =the 
magnification  of  the  optical  system  employed. 

Since  the  scale  on  the  micrometer  slide  is  divided  into  TJ^  mm.,  it  follows 
that 


Suppose,  for  instance,  the  distance  between  two  adjacent  lines  on  the 
paper  be  5  mm.  The  magnification  produced  will  be  100  x  5.  This  is 
expressed  by  saying  that  the  magnification  is  500,  or  to  be  more  accurate 
500  diameters. 

The  magnification  produced  by  an  optical  system  can  also  be  determined  by 
simply  projecting  the  magnified  divisions  of  the  micrometer  directly  on  to  a  milli- 
metre scale  arranged  on  the  same  level  as  the  microscope  stage. 

Then,  if  n  denote  the  number  of  divisions  on  the  scale  occupied  by  m  divisions 
on  the  micrometer, 

the  magnification  is  =  100    . 
m 


122  PRACTICAL  'DIRECTIONS 

Suppose  for  example  that  three  divisions  of  the  micrometer  occupy  fifteen 
divisions  on  the  millimetre  scale,  then  the  magnification  is  =  100  x  -V5  =500. 

This  is  a  simple  and  convenient  method  but  the  results  are  only  approxi- 
mate, the  magnification  being  somewhat  exaggerated. 

B.  With  the  ocular  micrometer.1  —  An  ocular  micrometer  consists  of  a  small 
circle  of  glass  on  which  a  scale  divided  into  —  mm.  is  engraved.  The  ocular 
micrometer  is  placed  between  the  eye  and  field  lenses  of  the  eyepiece. 

The  magnification  of  the  eyepiece  being  known  (generally  10  diameters), 
each  division  of  the  scale  as  seen  through  the  eyepiece  is  equal  to  -^  x  10 
mm.  =  1  mm. 

1.  Place  the  stage  micrometer  on  the  stage  of  the  microscope,  drop  the 
ocular  micrometer  into  the  eyepiece,  and  turn  on  the  objective  to  be  examined. 
Adjust  the  tube  to  the  proper  working  distance  and  focus  the  scale  on  the 
stage  micrometer,  then  arrange  the  latter  so  that  any  two  lines  on  it  coincide 
with  any  two  lines  on  the  ocular  micrometer. 

2.  Determine  how  many  divisions  of  the  ocular  micrometer  are  covered 
by  one  division  of  the  stage  micrometer,  and  let  n  be  the  number. 

The  magnification  produced  is  given  by 


and  if  five  divisions  of  the  ocular  micrometer  are  covered  by  one  division 
of  the  stage  micrometer,  the  magnification  is  =5  x  100. 

2.  The  measurements  of  objects  under  the  microscope. 

The  standard  adopted  for  microscopical  measurements  is  the  one-thousandth 
part  of  a  millimetre,  which  is  designated  by  the  Greek  letter  u  ;  the  Bacillus 
tuberculosis  for  example  is  said  to  measure  T7  to  3'5/*  long  by  0*2  to  0'5/x 
broad. 

Two  different  methods  may  be  employed  for  measuring  microscopical 
objects. 

A.  Camera  lucida  method.  —  1.  First  ascertain  the  magnifying  power  of 
the  system  of  lenses  to  be  used  by  means  of  the  objective  micrometer  and 
camera  lucida  (p.  121). 

2.  Substitute  the  slide  on  which  the  object  to  be  measured  is  mounted  for 
the  stage  micrometer,  and  an  outline  of  the  object  will  be  thrown  on  a  sheet 
of  paper  arranged  as  for  the  preceding  determination. 

3.  Measure  the  length  of  the  outline  in  millimetres,  and  let  n  be  the  length. 

4.  Then,  the  magnifying  power  m  of  the  combination  of  lenses  being  known, 
the  diameter  D  of  the  object  is  easily  determined  from  the  equation 


Let  us  suppose  the  magnifying  power  of  the  optical  system  to  be  500  diameters, 

[*  Hermann,  Whittaker,  and  Young  warn  against  the  use  of  an  Huygenian  eyepiece 
with  a  micrometer.  Ramsden's  is  the  only  eyepiece  that  can  be  relied  upon.  Clearly  a 
micrometer  scale  put  between  the  lenses  of  an  Huygenian  eyepiece  will  only  be  magnified 
by  the  eye  lens  and  will  therefore  undergo  "pincushion"  distortion.  But  the  image  of 
the  stage  micrometer  at  that  place  has  "barrel-shaped"  distortion  which  is  rectified  by 
the  "pincushion"  distortion  produced  by  the  eye  lens. 

[The  object  must  be  in  the  centre  of  the  field,  as  towards  the  periphery  "  pincushion  " 
distortion  would  be  more  marked. 

[Using  the  same  microscope  measurements  made  with  the  same  combination  of  lenses 
are  comparable  among  themselves  but  are  not  comparable  with  measurements  made 
with  any  other  microscope  nor  with  the  same  microscope  and  any  other  combination  of 
lenses.] 


MEASUREMENT   OF   OBJECTS  123 

and  the  greatest  diameter  of  the  outline  of  the  Bacillus  tuberculosis  as  sketched 
with  the  camera  lucida  to  be  T5  mm.  :    then,  from  the  formula 

D=J^  =0-003  mm.  =  3/4, 

we  find  the  length  of  the  tubercle  bacillus  to  be  3/4. 

A  table  can  be  readily  drawn  up  showing  the  magnification  obtained  with 
any  combination  of  lenses,  and  such  a  table  will  save  considerable  time  in 
the  measurement  of  microscopical  objects. 

B.  Measurement  with  the  ocular  micrometer  [see  footnote  p.  122]. — 1.  The 
stage  micrometer  is  examined  through  the  ocular  micrometer,  and  the  number 
of  divisions  on  the  ocular  micrometer  corresponding  to  one  on  the  stage 
micrometer  determined  for  each  objective.  For  example,  supposing  that  with 
objective  No.  8  one  division  of  the  stage  micrometer  cover  five  divisions  on 
the  ocular  micrometer,  then  five  divisions  on  the  ocular  micrometer  are 
equal  to  TJ^  mm.,  and  one  division  to  ^-^  mm.,  that  is  to  2/4. 

2.  Replace  the  stage  micrometer  by  the  object  to  be  measured.     Suppose 
it  occupies  n  divisions  on  the  scale. 

3.  Now,  knowing  that  one  division  is  equal  to  2/4,  and  using  D  to  denote 
the  diameter  of  the  object, 

T>=n  x2/4. 

If  the  object  cover  for  example  two  divisions,  then 

D=4/4. 

Note. — A  table  giving  the  value  of  each  division  of  the  ocular  micrometer  when 
used  with  any  objective  can  be  drawn  up.  It  is  then  only  necessary  to  multiply 
this  figure  by  the  number  of  divisions  occupied  by  an  object.  For  example,  using 
Reichert's  lenses — 

With  objective  No.  2  one  division  on  the  ocular  micrometer  scale  =21 /j. 
No.  4,          „  „  „  =11,,. 

No.  8,          „  „  „  =2-2/4. 

No.  9,          „  „  „  =l-9/x. 

„  i^sth,  „  „  „  =1*8/4. 

Thus  :  Suppose,  using  objective  No.  8  (Reichert),  an  object  covers  two  divisions 
on  the  ocular  micrometer  ;  then 

D  =2-2/xx2  =  4-4/4. 

Similarly,  an  object  seen  through  a  f^th  immersion  lens  covers  three  divisions  ; 
then  D  =  1  -8/4  x  3  =  5  '4/4. 

It  will  be  readily  understood  that  the  higher  the  magnification  the  more 
exact  the  measurement.  With  high  powers  the  errors  of  observation  are 
reduced. 


SECTION  VI.^DARK-GROUND   ILLUMINATION. 

It  has  already  been  shown  (p.  113)  that  it  is  impossible  even  with  the  best 
microscopes  to  distinguish,  i.e.  to  resolve,  any  two  points  less  than  about 
Opl/4  apart,  or  to  see  any  details  of  smaller  dimensions  than  0*1/4. 

To  render  small  delicate  objects  more  readily  visible  under  the  microscope, 
Siedentopf  and  Zsigmondy  have  utilized  the  fact  that  very  fine  particles 
placed  on  a  dark  back-ground  and  powerfully  illuminated  are  rendered  much 
more  easily  visible  than  when  examined  on  a  brightly  illuminated  surface. 
Everyone  is  familiar  with  tKis  fact  in  connexion  with  the  stars — the  darker 
the  night  the  brighter  the  stars.  This  is  the  whole  principle  of  [the  dark- 
ground  illuminator,  or,  as  it  sometimes  unfortunately  is  termed]  the  ultra- 
microscope.  The  dark-ground  illuminator  does  not  increase  the  resolving  power 


124 


DARK-GROUND  ILLUMINATION 


of  the  system  of  lenses,  but  merely  illuminates  particles  when  on  a  dark  back- 
ground and  so  renders  them  more  easily  visible. 

The  researches  of  Siedentopf  and  Zsigmondy,  afterwards  extended  by 
Cotton  and  Mouton,  have  been  taken  up  by  optical  instrument  makers,  who 
have  constructed  and  are  daily  improving  the  apparatus  necessary  for  dark- 
ground  illumination. 

1.  The  application  of  dark-ground  illumination 
to  micro-biology. 

Whatever  the  form  of  apparatus  employed,  the  dark-ground  illuminator 
does  not  appear  likely  to  be  of  assistance  in  the  study  of  infinitely  small 
things,  such  as  the  so-called  "invisible  micro-organisms,"  [for  the  simple 
reason  that  objects  less  than  0'1/x  are  not  resolved.  They  are  seen  just  as 
stars  are  seen,  which  subtend  no  appreciable  angle,  but  are  visible  because 
their  image  forms  such  an  intensely  bright  point  of  light  on  part  of  the  apex 
of  one  retinal  cone  that  they  become  visible.  Such  minute  objects  appear  as 
bright  points  in  the  field  of  vision  surrounded  by  light  and  dark  diffraction 
rings ;  they  have  neither  shape  nor  form.] 

The  instrument  is,  however,  of  considerable  practical  value  in  that  it  affords 
more  favourable  conditions  than  are  obtainable  with  the  ordinarily  illuminated 
microscope  stage  for  the  examination  of  material  in  the  fresh  unstained  con- 
dition. The  dark-ground  illuminator  renders  cells  and  organisms  easily 
visible  in  the  living  condition  with  their  natural  movements  unimpaired. 
The  valuable  aid  afforded  by  the  instrument  in  the  rapid  diagnosis  of  certain 
micro-organic  diseases,  and  particularly  of  syphilis,  has  been  demonstrated 
by  Landsteiner  and  Mucha,  by  Gastou  and  others. 

2.  The  construction  of  the  dark-ground  illuminator. 

The  essential  features  of  the  dark-ground  illuminator. — The  dark  back- 
ground and  the  powerful  illuminant  that  it  is  necessary  to  apply  can  be 
realized  in  several  ways. 

A.  Zeiss'  diaphragm. — The  simplest  and  cheapest  method — sufficient  more- 
over in  the  majority  of  cases  for  purposes  of  clinical  diagnosis — is  to  use  an 


FIG.  113. — Dark-ground  illuminator  for  fixing  below  the  stage. 


ordinary  microscope  fitted  with  an  Abbe  condenser  (N.A.  1'40),  a  dry  lens 
(7  or  8)  and  a  high  eyepiece  (Zeiss'  12  or  18  compensating  ocular)  :    the 


PRACTICAL   DETAILS  125 

apparatus  for  dark-ground  illumination  consists  of  a  special  diaphragm 
which  is  placed  below  the  condenser.  Slides  and  cover-glasses  of  a  given 
thickness,  varying  with  every  condenser,  are  essential. 

B.  Special  condensers. — In  these  cases  the  ordinary  Abbe  condenser  is 
replaced  by  a  prismatic  condenser  (Cotton  and  Mouton),  a  parabolic  con- 
denser (Zeiss)  or  a  spherical  condenser  (Leitz)  arranged  in  such  a  way  that 
the  rays  reflected  by  the  mirror  are  deviated,  so  that  they  pass  obliquely 
through  the  film  of  liquid  which  is  placed  between  the  slide  and  cover-glass, 
and  cannot  enter  the  objective.  Under  these  conditions  any  particles  held 
in  suspension  in  the  preparation  on  the  stage  of  the  microscope  are  lighted 
from  the  sides  while  the  back-ground  is  obscure. 

In  most  patterns  the  dark-ground  condenser  is  placed  below  the  stage 


FIG.  114. — Dark-ground  illuminator  for  fixing  on  the  stage. 

in  the  collar  generally  used  for  the  Abbe  condenser,  but  instruments  are  now 
made  to  fix  on  the  stage  of  the  microscope. 

These  latter  are  the  better,  and  they  can  be  used  either  with  a  dry  lens  or 
with  an  immersion  lens. 

3.  Method  of  using  the  dark-ground  illuminator. 

To  use  dark-ground  illumination  it  is  necessary  to  have  : 

1.  A  powerful  source  of  light ; 

2.  A  lens  to  form  the  image  of  this  source  on  the  mirror  ; 

3.  A  firm  microscope  stage  on  which  to  fix  the  dark-ground  illuminator,  an 
objective  and  an  eyepiece. 

These  are  all  arranged  on  a  rigid  table,  and  it  is  an  advantage  to  have  an 
optical  bench  1  metre  long. 

A.  The  source  of  light. 

The  specific  intensity  of  the  light  increases  the  visibility  of  the  objects 
under  the  microscope.  A  Nernst  lamp,  an  arc  lamp  or  an  inverted  incan- 
descent gas  burner  are  the  sources  of  light  generally  used.  Electric  light  is 
perhaps  better,  but  an  Auer  burner  (inverted  incandescent)  (p.  118)  is  quite 
good  enough  for  most  purposes. 

Sometimes  it  is  necessary  to  use  sunlight,  and  particularly  when  photo- 
graphing objects  under  the  ultra-microscope.  For  this  purpose  the  apparatus 
is  arranged  in  a  dark  chamber,  and  the  rays  of  light  falling  on  an  heliostat 
worked  by  clock-work  pass  into  the  chamber  through  an  opening  made  in 
the  shutter  of  the  window. 

Whatever  the  pattern  of  apparatus  used,  the  rays  of  light  must  be 
condensed  by  a  lens  on  to  the  flat  surface  of  the  microscope  mirror. 

Sometimes  it  is  better  to  use  instead  of  a  lens  a  large  round  flask  filled 


126  DARK-GROUND   ILLUMINATION 

with  water  lightly  tinted  with  copper  sulphate,  an  arrangement  which  has 
the  advantage  of  absorbing  the  heat  rays  and  so  prevents  deterioration  of 
the  preparation  from  that  cause. 

The  image  of  the  source  of  light  must  be  formed  on  the  mirror  :    to  secure 
this,  a  sheet  of  white  paper  may  be  placed  upon  the  surface  of  the  mirror 


FlG.  115. — Illumination  with  Nernst  lamp  and  lens. 

which  is  then  moved  about  until  the  image  is  clearly  defined.  The  mirror 
should  be  uniformly  illuminated  and  the  whole  surface  covered  with  light- 
To  get  the  light  arranged  satisfactorily  requires  prolonged  manipulation, 
so  that  for  clinical  work  where  time  is  an  important  consideration  the 
apparatus  should  be  arranged  beforehand.  It  is  of  great  advantage  in  this 
connexion  to  have  an  optical  bench,  for  with  it  the  respective  positions  of 
the  light,  the  lens  and  the  microscope  can  be  found  once  for  all.  Roughly 
speaking,  the  lamp,  the  lens  and  the  mirror  are  placed  at  a  distance  of  15  to 
20  cm.  from  one  another  according  to  the  apparatus  used.  The  tube  of  the 
microscope  should  be  vertical. 

B.  Centering. 

The  dark-ground  illuminator,  whether  placed  on  the  stage  of  the  microscope 
or  arranged  in  the  place  of  the  Abbe  condenser,  must  be  centered.  The  method 
by  which  this  is  done  will  depend  upon  whether  the  apparatus  is  above  or 
below  the  stage. 

(a)  Dark-ground  illuminators  fixed  in  the  collar  ordinarily  carrying  the 
Abbe  condenser  must  be  so  arranged  that  the  lower  flange  is  close  up  against 
the  collar,  and  the  upper  surface  just  below  the  upper  surface  of  the  stage. 

Using  a  low-power  objective  and  looking  down  the  tube  of  the  microscope, 
the  centre  of  the  apparatus  should  be  brightly  illuminated  without  shadows 
or  halos.  If  the  field  be  not  bright,  adjust  the  lateral  screws  (fig.  113)  until 
the  lighting  appears  quite  uniform. 

(b)  In  those  forms  which  are  made  for  use  on  the  stage,  first  fix  the  apparatus 
with  the  clips  and  then,  using  a  low-power  objective  and  looking  down  the 
tube,  take  hold  of  it  on  each  side  with  thumb  and  finger  and  move  it  about 
gently  until  the  centre  appears  brightly  and  uniformly  illuminated. 


PRACTICAL   DETAILS  127 

C.  Arrangement  of  the  preparation  to  be  examined. 

1.  The  preparation  to   be  examined  should  be  mounted  on  a  slide  and 
covered  with  a  cover-glass. 

(a)  The  slide  should   be   of  crystal   glass  free  from  flaws  and  absolutely 
clean,    because   any   dust    or    dirt   will    seriously   interfere   with    the    ob- 
servation. 

Slides  and  cover-glasses  should  be  washed  in  acid  rinsed  in  distilled  water 
and  kept  in  alcohol  (p.  130).  When  required  for  use,  it  is  advisable  in  order 
to  ensure  cleanliness  to  paint  the  slide  with  a  layer  of  collodion,  which  can 
be  peeled  off  just  before  it  dries. 

Dust  which  falls  on  the  cover-glass  during  the  examination  interferes 
with  the  satisfactory  lighting  of  the  preparation,  and  if  the  observation 
be  prolonged  the  cover-glass  should  be  washed  or  dusted  from  time  to 
time. 

(b)  To  secure  the  most  satisfactory  illumination  the  slide  should  be  of  a 
thickness  suitable  to  the  particular  apparatus  in  use  (all  dark-ground  con- 
densers are  marked  with  a  number  indicating,  the  thickness  of  slide  to  be 
used — generally  about  1*4  mm.).     When  working  with  sunlight  it  is  absolutely 
necessary  that  slides  of  the  exact  thickness  indicated  on  the  condenser  should 
be  used  ;   but  with  the  sources  of  light  ordinarily  employed  this  precision  is 
of  less  importance,  and  one-third  of  a  millimetre  one  way  or  the  other  is  a 
matter  of  no  great  moment. 

The  thickness  of  the  cover-glasses  should  correspond  with  the  correction 
of  the  objective  (p.  119). 

2.  There  should  be  continuity  between  the  media  through  which  the  light 
passes,  so  that  refraction  may  take  place  under  the  best  conditions  ;   a  large 
drop  of  very  fluid  immersion  oil  should  therefore  be  placed  between  the 
condenser  and  the  slide. 

An  inferior  quality  of  oil  is  a  frequent  cause  of  failure.  The  oil  should  be  quite 
fluid,  absolutely  homogeneous,  contain  no  air  bubbles,  and  be  used  in  sufficient 
quantity  to  completely  fill  the  space  between  the  lens  and  the  condenser. 

3.  The  film  to  be  examined  should  be  as  thin  as  possible,  uniform  and  free 
from  air  bubbles.     If  the  material  be  sufficiently  fluid  and  viscous  to  keep  the 
slide  and  cover-glass  together  the  preparation  may  be  examined  without  any 
addition.     In  the  contrary  case,  dilute  the  material  in  a  drop  of  blood  serum, 
aqueous  humour  or  ascitic  fluid ;  water  or  normal  saline  solution  may  be 
used  but  these  solutions  have  the  disadvantage  that  they  alter  the  shape 
and  interfere  with  the  vitality  of  the  cells. 

If  the  experiment  is  to  be  prolonged  it  is  advisable  to  lute  the  edge  of  the 
cover-glass  with  a  little  vaseline  or  paraffin  to  prevent  evaporation. 

D.  Focussing  the  microscope. 

For  dark-ground  illumination  work  a  dry  lens  (No.  7,  8,  or  9)  may  be  used 
(though  an  immersion  lens  is  better)  and  a  high  eyepiece  (No.  IV.  or  Zeiss' 
compensating  ocular  18). 

To  obtain  a  quite  black  background,  special  objectives  can  be  employed  in  the 
mounting  of  which  a  carefully  centered  diaphragm  is  suspended  to  intercept  marginal 
rays :  these  objectives  (Leitz,  Zeiss)  give  remarkably  distinct  images. 

A  certain  amount  of  skill  which  can  only  be  obtained  with  practice  is 
required  to  get  satisfactory  results. 

1.  With  a  dry  objective.— The  lighting  being  satisfactory,  the  apparatus 
centered  and  the  preparation  fixed  with  the  clips,  the  eye  is  applied  to  the 
tube  of  the  microscope  which  is  then  slowly  lowered.  At  first  there  is  a 


128  DARK-GROUND   ILLUMINATION 

certain  amount  of  diffused  light,  but  this  soon  gives  place  to  complete  dark- 
ness ;  by  continuing  carefully  to  lower  the  tube,  the  back-ground  will  suddenly 
become  lit  up  in  places  and  dotted  with  bright  points  ;  the  preparation  is 
then  focussed. 

2.  With  an  oil-immersion  lens. — Place  a  drop  of  cedar-wood  oil  on  the 
cover-glass  and  lower  the  tube  until  the  lens  touches  the  oil.  Then  with  the 
mechanical  adjustment  gently  raise  and  lower  the  tube  until  the  back-ground 
is  illuminated  with  bright  spots. 

If  the  field  be  unequally  lighted  or  if  it  be  narrowed  by  shadows,  the 
centering  is  at  fault  and  must  be  corrected  by  careful  manipulation  of  the 
dark-ground  condenser  (p.  126). 

E.  Appearances  seen  in  the  field  under  dark-ground  illumination. 

When  the  lighting  and  centering  are  satisfactory,  and  the  object  focussed, 
luminous  points  and  spots  of  different  appearances — motile  or  non-motile — 
will  be  seen  corresponding  to  the  microscopical  objects  (micro-organisms, 
cells,  particles  of  colloid  matter,  etc.)  in  the  preparation.  Certain  non-motile 


FlG.  116. — Preparation  showing  spirochaetes,  leucocytes  and  red  cells  (after  Gastou). 

spots,  generally  taking  the  form  of  rosettes  or  flocculent  masses,  may  be 
seen  ;  these  are  merely  flaws  in  the  glass  and  must  not  be  confused  with 
the  objects  in  the  preparation.  [This  generalization  of  course  only  applies 
when  the  size  of  the  objects  is  greater  than  the  resolving  power  of  the  com- 
bination of  lenses  employed.  Any  objects  in  the  field  which  are  beyond  the 
resolving  power  of  the  combination  of  lenses  will  appear  as  bright  spots 
with  light  and  dark  diffraction  rings  and  the  size  of  the  objects  which  will 
appear  as  such  will  depend  upon  the  intensity  of  the  illumination.  It  has 
already  been  pointed  out  that  the  so-called  ultra-microscope  or  dark-ground 
illuminator  does  not  increase  the  resolving  power  of  the  microscope,  hence 
whatever  the  shape  of  the  object  if  it  be  so  small  as  to  be  below  the  resolving 
power  of  the  system  of  lenses  used  it  will  appear  as  a  bright  dot  surrounded 
by  rings.] 

It  will  be  found  easy  to  study  the  movements  (Brownian  movements,  move- 
ments of  propulsion,  etc.)  of  the  different  corpuscles.  In  interpreting  these 
it  must  not  be  forgotten  that  an  universal  movement  of  the  illuminated 


PRACTICAL  DETAILS 


129 


elements   in    the    same   direction   is   due   to   currents   set   up   in   the   pre- 
paration. 


FIG.  117. — Preparation  showing  red  blood  cells,  hsematoblasts  and  strandg 
of  fibrin  (after  Gastou). 

Lastly  it  cannot  be  too  strongly  emphasized  that  the  smallest  trace  of  dust 
on  the  slides  or  cover-glasses  interferes  materially  with  the  examination 
of  the  preparation. 


CHAPTER  VIII. 

THE  MICROSCOPICAL  EXAMINATION  OF  CULTURES 
OF  MICRO-ORGANISMS. 

Introduction. 

Section  I.— The  preparation  of  slides  and  cover-glasses,  p.  130. 
Section  II. — The  examination  of  unstained  preparations,  p.  131. 
Section  III. — The  examination  of  stained  preparations,  p.  135. 

1.  Staining  solutions,  p.  137.     2.  Simple  staining,  p.  140.     3.  Gram's  stain,  p.  142. 
4.  Claudius'  method,  p.  144. 

CULTURES  should  be  examined  microscopically  in  two  ways. 

(a)  An  unstained  preparation  of  the  living  organisms  should  first  be 
examined.  By  this  means  not  only  can  the  shape  of  the  organisms  be 
determined  but  also  whether  they  are  motile  or  not,  and  if  motile  the  nature 
and  rapidity  of  the  movements. 

(6)  Secondly,  the  morphological  study  of  an  organism  must  be  completed 
by  the  examination  of  stained  preparations,  which  will  allow  a  more  detailed 
study  of  its  structure  with  the  higher  powers  of  the  microscope. 

For  the  preparation  of  objects  for  the  microscope  a  supply  of  clean  slides 
and  cover-glasses  is  essential,  and  the  methods  of  preparing  these  may  first 
be  described. 


SECTION  I.— THE   PREPARATION   OF   COVER-GLASSES   AND   SLIDES. 

The  essential  qualities  of  cover-glasses  and  slides  have  already  been  men- 
tioned (p.  119).  Before  being  used  they  must  be  carefully  cleaned. 

1.  Cleaning  of  cover-glasses  and  slides. 

A.  New  cover-glasses  are  more  or  less  greasy  and  cannot  be  moistened 
with  water.  Before  using  them  therefore  wash  them  in  95  per  cent,  alcohol, 
and  wipe  with  a  piece  of  soft  smooth-surfaced  cloth ;  then  to  get  them 
perfectly  clean  they  must  be  passed  several  times  through  the  heating  flame 
of  a  Bunsen  burner. 

In  wiping  a  cover-glass  never  hold  it  in  both  hands  because  it  will  certainly 
be  broken,  but  hold  it  between  the  folds  of  the  cloth  with  the  thumb  and 
first  finger  of  the  right  hand,  and  rub  it  gently. 

It  is  convenient  to  have  a  wide-mouthed  ground-glass  stoppered  pot  on 
the  bench  containing  95  per  cent,  alcohol  in  which  to  keep  a  stock  of  cover- 
glasses,  so  that  they  can  be  taken  out  and  dried  as  wanted. 


SLIDES  AND   COVER-GLASSES  131 

Slides  similarly  should  be  carefully  washed  in  alcohol  and  dried. 

B.  Slides  and  cover-glasses  can  be  used  over  and  over  again.  They  must 
however  be  carefully  cleaned  to  remove  all  traces  of  material  on  them  ; 
unless  this  be  properly  done  mistakes  are  likely  to  occur  when  they  are  used 
a  second  time.  The  thorough  cleaning  of  soiled  slides  is  therefore  of  great 
importance  and  can  be  done  as  follows  : 

1.  Drop  all  used  slides  and  cover-glasses  when  they  are  finished  with  into 
a  dish  containing  spirit. 

2.  When  a  number  have  collected  put  them  into  a  porcelain  dish,  cover 
them  with  a  4  per  cent,  solution  of  sodium  carbonate  and  boil  for  half  an 
hour. 

3.  Pour  off  the  soda  solution,  wash  in  a  large  volume  of  water,  then  drop 
them  into  the  following  solution  : 

Water,  -  -     1000  grams. 

Potassium  bichromate,     -  -         50       „ 

Sulphuric  acid,        -  -       100      „ 

and  boil  again  for  half  an  hour. 

4.  Pour  off  the  bichromate  solution,  wash  again  in  a  large  volume  of  tap 
water,  then  in  distilled  water,  wipe  them  dry  and  drop  them  one  by  one  into 
covered  pots  filled  with  95  per  cent,  alcohol,  out  of  which  they  can  be  taken 
as  required. 

This  method  will  ensure  the  glasses  being  clean. 

2.  Method  of  using  cover-glasses  and  slides. 

Cover-glasses  should  be  picked  up  by  one  of  their  angles  with  a  pair  of 
Cornet's  (fig.  118)  or  Debrand's  (fig.  119)  forceps. 


FIG.  118. — Cornet's  forceps. 


Debrand's  forceps,  a  very  useful  modification  of  Cornet's,  are  well  balanced 
and  easily  held  in  the  hand  :    they  give  a  firm  hold  and  do  not  break  the 

cover-glasses. 


FIG.  119. — Debrand's  forceps. 


SECTION   II.— THE    EXAMINATION    OF   UNSTAINED    PREPARATIONS. 

A  little  drop  of  a  culture  of  a  micro-organism  may  be  mounted  between  a 
slide  and  cover-glass  and  examined.  But  to  keep  the  organisms  alive  while 
they  are  being  examined  for  the  purpose  of  studying  the  method  of  multiplica- 
tion, etc.,  special  slides  having  a  small  concavity  or  cell  ground  in  their  centre, 
are  used.  A  drop  of  broth  is  placed  in  the  cell  and  sown  with  the  organism ; 
in  this  way  a  living  culture  is  available  for  the  purposes  of  microscopical 
examination. 


132  UNSTAINED   PREPARATIONS 


1.  Examination  of  a  culture  on  an  ordinary  slide. 

A.  Cultures  in  fluid  media. — 1.  Prepare   an  absolutely  clean  slide  and 
cover-glass. 

2.  Aspirate  a  few  drops  of  the  culture  into  a  Pasteur  pipette,  taking  care 
of  course  not  to  introduce  contaminations. 

3.  Pick  up  a  cover-glass  by  one  of  its  corners  with  a  pair  of  Cornet's  forceps, 
and  let  fall  a  drop  of  the  liquid  from  the  pipette  on  to  the  centre  of  the  cover- 
glass. 

4.  Invert  the  cover-glass  on  to  a  slide  and  the  drop  will  spread  out  in  a 
thin  layer.     One  must  be  careful  not  to  introduce  any  air  bubbles  as  these 
would  interfere  with  the  subsequent  examination. 

5.  Place  the  preparation  on  the  stage  of  the  microscope  and  examine  with 
a  No.  8  or  No.  9  objective  and  a  No.  I.  or  No.  II.  eyepiece.    If  the  examination 
is  likely  to  be  prolonged  the  edges  of  the   cover-glass  can  be  luted  with 
paraffin  in  the  following  manner :  Soak  up  the  excess  of  culture  fluid  which 
has  exuded  from  the  edges  of  the  cover-glass  with  a  cigarette  paper  or  piece 
of  filter  paper  :    then  apply  a  heated  iron  rod — it  is  better  to  use  a  special 
instrument  such  as  that  shown  in  fig.  120 — to  a  block  of  paraffin,  so  as  to 


FIG.  120. — Instrument  for  luting  with  paraffin. 

melt  a  little  of  it :  in  doing  this  some  of  the  paraffin  will  adhere  to  the  rod 
and  can  be  transferred  to  each  of  the  corners  of  the  cover-glass  to  fix  it  in 
position.  Then  by  taking  up  some  more  paraffin  on  the  rod  the  edges  can 
be  luted. 

The  pipette  with  which  the  culture  was  removed  should  not  be  used  again. 
Pipettes  which  have  been  in  contact  with  a  culture  must  never  on  any  account 
be  laid  on  the  bench.  All  pipettes  after  use  should  be  put  into  a  metal  vessel, 
and  when  the  experiment  in  hand  is  completed  sterilized  either  in  the  autoclave 
or  more  readily  by  boiling  for  a  few  minutes  :  only  then  can  they  be  safely  thrown 
away. 

B.  Cultures  on  solid  media. — 1.  Take  a  cover-glass  in  a  pair  of  forceps,  and 
put  a  little  drop  of  recently  filtered  water  (Chamberland  filter)  or  sterile 
broth  in  its  centre. 

2.  Open  the  culture-tube  in  the  ordinary  way,  take  up  a  trace  of  the  culture 
on  a  platinum  wire  and  re-plug  the  tube. 

3.  Make  an  emulsion  of  the  culture  in  the  drop  of  water  on  the  cover-glass 
with  the  wire.     Flame  the  wire. 

4  and  5.  As  above. 

A  common  mistake  is  to  remove  too  much  of  the  culture.  If  more  than  a  trace 
be  taken,  there  will  be  too  many  organisms  in  the  field  of  the  microscope  and'  the 
examination  of  them  will  be  exceedingly  difficult.  It  cannot  be  too  clearly  under- 
stood that  the  fewer  the  organisms  the  better  can  their  shape,  movements,  etc., 
be  studied. 

2.  Hanging  drop  preparations. 

By  using  a  hollow-ground  slide  any  organism  under  examination  can  be 
kept  alive  for  a  long  time  and  its  development  studied. 

(i)   The  technique  of  the  hollow-ground  slide. 

There  are  many  patterns  of  slides  or  cells  for  use  with  the  microscope. 
A.  Koch's  hollow-ground  slide. — This  is  simply  a  slide  of  the  ordinary  size 


HANGING  DROP  PREPARATIONS  133 

having  a  circular  cup-shaped  hollow  about  15  mm.  in  diameter  ground  in 
its  centre  (fig.  121).     Sterilize  the  slide  as  well  as  the  cover-glass  with  which 


FIG.  121.— Koch's  hollow-ground  slide. 

it  is  to  be  covered  by  rapidly  passing  them  through  the  flame  several  times 
just  before  they  are  about  to  be  used. 

(a)  In  the  case  of  cultures  already  incubated,  take  a  drop  of  the  culture 
and  place  it  in  the  centre  of  the  previously  heated  and  cooled  cover-glass, 
invert  the  cover-glass  over  the  hollow  in  the  slide  and  ring  the  edges  with  a 
little  vaseline  to  prevent  evaporation.     The  drop  of  culture  hangs  from  the 
lower  surface  of  the  cover-glass  into  the  cavity  ground  in  the  slide. 

The  drop  of  culture  placed  on  the  cover-glass  should  be  small  enough  to  prevent 
it  touching  the  sides  of  the  cavity  otherwise  the  liquid  will  run  by  capillarity  between 
the  slide  and  cover-glass  and  the  hanging  drop  will  disappear. 

When  examining  a  hanging  drop  under  the  microscope  great  care  must  be  exer- 
cised in  lowering  the  tube,  because  the  cover-glass  is  only  supported  at  its  edges 
and  the  least  pressure  on  it  will  break  it.  It  is  best  to  use  a  No.  8  or  No.  9  objec- 
tive and  No.  I.  or  No.  II.  eyepiece  (Reichert's  lenses). 

The  small  quantity  of  air  contained  within  the  cell  is  quite  sufficient  to  provide 
all  the  oxygen  necessary  for  several  days. 

(b)  Most  frequently  a  hanging  drop  is  used  to  study  the  development  of 
an  organism.     In  this  case  the  culture  must  be  sown  in  the  cell.     It  can  be 
done  thus :    Put  a  drop  of  sterile  broth  or  sterile  aqueous  humour  on  the 
cover-glass  and  sow  it  with  the  organism  under  investigation. 

It  is  absolutely  essential  in  doing  this  that  only  a  very  few  organisms  be  sown. 
A  trace  of  the  culture  may  be  picked  up  on  the  end  of  the  straight  wire  and  the 
drop  then  very  lightly  touched  with  the  latter,  but  it  is  better  to  adopt  the  dilution 
method  :  thus,  sow  a  broth  tube  (No.  1)  with  a  loopful  of  the  culture  and  shake; 
sow  a  second  broth  tube  (No.  2)  with  one  or  perhaps  two  drops  from  tube  No.  1, 
and  then  transfer  a  drop  of  the  broth  from  tube  No.  2  to  the  cover-glass  to  form 
the  hanging  drop.  If  tube  No.  2  still  contain  too  many  organisms,  sow  a  third 
tube  (No.  3)  with  a  few  drops  from  No.  2.  The  hanging  drop  is  then  made  with  a 
drop  of  broth  from  No.  3. 

The  successive  steps,  then,  are  as  follows  : 

1.  Flame  the  slide  and  cover-glass  and  allow  them  to  cool. 

2.  Place  a  drop  of  sterile  broth  in  the  centre  of  the  cover-glass  and  sow  it 
with  a  trace  of  the  culture  (or,  better,  take  a  drop  of  broth  from  a  tube  sown 
by  the  dilution  method). 

3.  Invert  the  cover-glass  on  the  hollow-ground  slide  and  lute  the  edges 
with  paraffin. 

4.  Examine  the  hanging  drop  on  a  warm  stage  (vide  post),  or  if  a  warm 
stage  be  not  available,  incubate  it  in  the  ordinary  incubator  and  examine 
at  frequent  intervals  on  the  ordinary  stage,  using  a  No.  8  or  No.  9  objective 
and  a  No.  I.  or  No.  II.  eyepiece.     Make  certain  that  at  the  time  when  the 
hanging  drop  is  made  there  are  not  more  than  two  or  three  organisms  in  each 
field  of  the  microscope. 

The  culture  can  be  kept  for  examination  for  1  to  3  days.     The  air  present 


134  UNSTAINED   PREPARATIONS 

in  the  cell  is  generally  quite  sufficient  for  the  growth  of  the  organism  during 
this  period. 

To  improvize  a  hollow-ground  slide. — A  hollow-ground  slide  may  be  improvized 
by  taking  a  rectangular  piece  of  pasteboard  about  3x2  cm.  and  1  '5  to  2  mm.  thick, 
and  cutting  out  of  its  centre  a  small  piece  about  15  mm.  square.  Sterilize  the  piece 
of  pasteboard  in  the  autoclave  at  115°  C.,  take  it  out  with  a  pair  of  sterile  forceps 
and  lay  it  on  a  slide  which  has  been  passed  through  the  flame:  the  cover-glass 
on  which  the  drop  of  fluid  is  placed  can  be  inverted  on  this  to  form  a  hanging  drop. 

B.  Bcettcher's  cell. — This  cell  consists  of  a  glass  slide  on  to  which  a  glass 
ring  (15-20  mm.  in  diameter  and  5  mm.  deep)  is  stuck  (fig.  122).  The  cover- 


FIG.  122.— Bcettcher's  cell. 

glass  carrying  the  hanging  drop  is  inverted  on  to  the  ring.  A  little  drop  of 
water  should  be  put  in  the  bottom  of  the  cell  to  prevent  evaporation  of  the 
culture  medium. 

C.  Ranvier's  cell. — In  the  foregoing  cells  the  hanging  drop  has  a  spherical 
lower  surface,  with  the  result  that  the  rays  of  light  passing  through  it  are 
refracted  at  points  which  are  not  equally  distant  from  the  lens,  and  this  to 
some  extent  interferes  with  the  examination  of  the  preparation.  For  delicate 
work  it  is  better  to  have  the  two  surfaces  of  the  liquid  under  examination 
parallel  to  each  other.  This  can  be  attained  by  using  Ranvier's  cell  (fig.  123), 


FIG.  123. — Ranvier's  cell. 

consists  of  a  rather  thick  glass  slide  having  a  circular  groove  15-20  mm. 
in  diameter  running  round  its  centre  marking  off  a  central  elevation  which 
it  surrounds  on  all  sides  like  a  moat.  The  upper  surface  of  this  elevated 
central  part  is  about  TVth  mm.  below  the  surface  of  the  slide.  The  drop  of 
liquid,  being  placed  on  the  central  elevation  and  covered  with  a  cover-glass, 
is  flattened  out  between  the  elevated  part  and  the  cover-glass,  and  forms  a 
layer  T^th  mm.  deep  surrounded  on  all  sides  by  the  air  in  the  groove  ;  the 
edges  are  luted  and  the  subsequent  procedure  is  the  same  as  in  the  foregoing 
cases. 

(ii)  The  cultivation  and  preservation  of  hanging  drop  preparations. 

To  grow  an  organism  under  these  conditions  it  is  necessary  to  keep  it  at 
the  temperature  best  suited  to  its  growth,  which  in  the  majority  of  cases  is 
37°  C.  This  may  be  done  by  keeping  the  slide  in  the  incubator,  taking  it 
out  when  required  for  microscopical  examination  ;  but  it  is  better  to  maintain 
the  slide  at  the  temperature  required  on  the  stage  of  the  microscope  itself, 


HANGING   DROP  PREPARATIONS 


135 


by  making  use  of  some  form  of  warm  stage  for  the  purpose,  VignaFs  for 
example  (fig.  124)  or  Malassez's  or  Ranvier's.     These  really  are  small  incu- 


FIG.  124. — Vignal's  warm  stage. 

bators,  allowing  of  the  examination  of  the  culture  through  a  circular  aperture 
cut  in  the  apparatus. 

Pfeiffer's  warm  stage  is  simpler  than  those  already  mentioned  and  serves 
the  same  purpose.  It  consists  of  a  rectangular  glass  box  (fig.  125A),  the  upper 
surface  of  which  is  hollowed  out  to  form  a  cell,  in  which  the  culture  is  placed. 
The  box  is  filled  with  water  and  is  connected  by  means  of  two  lateral  tubulures 
to  a  thermostat.  The  temperature  is  indicated  by  a  thermometer  placed  as 
shown  in  the  figure. 


FIG.  125A.— Pfeiffer's  warm 


FIG.  125B. — Pfeiffer's  stage  in  section. 

The  apparatus  is  placed  on  the  stage  of  the  microscope  like  an  ordinary 
slide. 

By  another  method  the  lower  part  of  the  microscope  is  enclosed  in  a  box — 
a  small  incubator — which  entirely  surrounds  the  stand  ;  the  box  has  a  window 
for  lighting  purposes  and  lateral  openings  to  allow  of  the  preparation  being 
moved  (Zeiss,  Plehn).  The  apparatus  is  fitted  with  a  regulator  and  is 
heated  by  a  gas  burner.  The  temperature  must  not  exceed  45°  C.,  to  avoid 
injury  to  the  microscope. 


SECTION   III.— THE   EXAMINATION   OF  STAINED  PREPARATIONS. 

Staining  methods  allow  a  more  detailed  study  of  the  morphology  of  micro- 
organisms than  is  possible  with  unstained  preparations,  and  furnish  important 
data  for  the  diagnosis  of  species.  For  different  species  of  bacteria  do  not 
react  in  the  same  way  to  stains  :  some  are  readily  stained  and  cannot  be 
decolourized  with  alcohol,  others  which  stain  with  equal  readiness  lose  the 


136  STAINED   PREPARATIONS 

stain  in  alcohol,  while  a  third  group  stain  with  difficulty  but  after  being 
stained  resist  the  action  of  the  most  powerful  decolourizing  agents. 

Bacteria  are  vegetable  cells  of  which  the  greater  part  is  occupied  by  the 
nucleus  (Biitschli)  :  they  stain  with  those  dyes  which  stain  the  nuclei  of 
vegetable  cells,  that  is  to  say,  the  basic  aniline  dyes. 

Stains. — Ehrlich  divided  dyes  according  to  their  action  on  cells  into  two 
groups  :  basic  dyes  and  acid  dyes. 

Basic  dyes  are  those  in  which  the  staining  property  depends  upon  a  base 
combined  with  a  colourless  acid.  They  are  called  selective  dyes,  because 
they  exhibit  a  marked  selective  affinity  for  nuclei  and  especially  the  nuclei 
of  vegetable  cells.  The  basic  dyes  are  the  true  micro-organic  dyes.  Those 
most  commonly  used  are  the  following  : 


Violets,  - 


Crystal-violet. 

Thionin  (Lauth's  violet). 

Gentian-violet. 

Methyl-violet  B  (Bale's  violet). 


Methyl- violet  6B. 
Paris  violet. 
Dahlia. 

Methylene  blue. 
Victoria  blue. 
Azur. 

ies'    '  Nile  blue,  or  Capri's  blue. 

Quinoline  blue. 
Unna's  polychrome  blue. 

I  Fuchsin. 
I  Rubin. 
I  Safranin. 
I  Neutral-red. 

(Methyl- green. 
\Malachite  green. 

Bismarck  brown,     -       Vesuvin. 
Colin  black,    -          -       Indulin. 

In  the  acid  dyes  on  the  other  hand  the  staining  agent  is  an  acid  combined 
with  a  coloured  or  colourless  base.  They  are  non-selective  dyes  and  stain 
all  tissues  indifferently .  Fluorescein  (phthalic  ether  of  resorcin),  eosin 
(tetrabrom-fluorescein),  aurantia,  coccinine,  acid  fuchsin,  tropseolin,  magenta 
8,  orange  Gr,  and  picro-carmine  are  the  acid  dyes  in  most  common  use. 

Note. — The  aniline  dyes  have  intense  staining  properties,  and  should  be  carefully 
handled;  if  the  hands  be  stained  accidentally  they  can  be  quite  easily  decolourized 
with  soap.  The  powders  should  not  be  shaken. 

Mordants.— In  dyeing,  an  intermediary  agent  is  used  to  fix  the  dye  more 
firmly  in  the  fabric.  This  intermediary  agent  is  known  as  a  mordant,  and 
combining  both  with  the  dye  and  with  the  tissue  unites  the  two  intimately 
together. 

Mordants  are  also  used  in  staining  micro-organisms,  and  though  their 
mode  of  action  is  not  as  yet  thoroughly  understood,  they  undoubtedly  increase 
the  affinity  of  the  dyes  for  the  cells  and  render  the  staining  more  rapid  and 
more  lasting.  The  mordants  in  ordinary  use  are  : 

Acids. — Acetic  acid. 

Phenol. — Creosote. 

Tannin. 

Iodine  in  iodine-iodide  solution. 

Bromine  in  iodine- bromide  and  bromine- bromide  solutions. 

Perchloride  of  mercury. 


STAINING  SOLUTIONS  137 

Alkalis. — Caustic  potash,  ammonia,  sodium  borate,  ammonium  carbonate,  and  certain 
organic  alkalis  (aniline,  phenylamine,  toluidine). 

Mixtures  of  two  dyes,  of  which  one  acts  as  a  mordant  towards  the  other. 

Action  of  heat. — The  rapidity  and  depth  of  the  staining  can  be  increased 
by  heating  the  preparation  in  a  bath  of  the  stain  to  60°  or  100°  C. 

1.  Staining-  solutions. 

The  staining  solutions  used  in  bacteriology  are  very  numerous.  Every 
observer  has  his  own  preferences,  so  that  there  is  a  multiplicity  of  formulae, 
making  the  subject  very  complicated  and  embarrassing  for  the  beginner  and 
practical  work  would  gain  much  by  a  reduction  and  simplification  of  these 
staining  processes.  As  a  matter  of  fact  a  few  formulae  will  meet  all  ordinary 
requirements,  and  if  these  be  thoroughly  understood  errors  which  often  arise 
from  the  use  of  too  complicated  and  unfamiliar  methods  will  be  avoided. 

The  various  formulae  to  be  found  in  papers  published  during  recent  years  must 
be  given,  but  those  methods  which  in  our  own  experience  have  given  good  results 
will  be  distinctly  indicated  and  will  be  found  sufficient  for  practically  all  purposes. 
The  acid  dyes  will  not  be  dealt  with  in  this  chapter  but  will  be  referred  to  later, 
and  the  consideration  of  some  of  the  staining  methods  of  limited  application  will 
be  deferred  until  occasion  for  their  use  arises. 

To  avoid  mistakes  only  good  dyes  obtained  from  well-known  sources 
should  be  used. 

A.  Simple  solutions. 

These  solutions  have  only  a  limited  use  ;  staining  solutions  containing  a 
mordant  are  generally  better. 

(i)   Alcoholic  solutions. 

Alcoholic  solutions  of  the  basic  aniline  dyes  are  prepared  by  mixing  in  a 
ground-glass  stoppered  bottle  : 

Dye,       -  1  gram. 

Absolute  alcohol,     -  -         10  c.c. 

Shake  well  and  leave  the  alcohol  standing  on  the  dye.  Filter  before  use. 
Alcoholic  solutions  keep  for  a  very  long  time  in  the  dark,  and  solutions  of  the 
following  dyes  should  be  kept  in  the  laboratory,  viz.  fuchsin,  crystal- violet 
or  gentian-violet,  and  methylene  blue. 

These  solutions  are  not  used  for  staining,  but  when  diluted  with  water 
serve  for  the  preparation  of  watery  alcoholic  solutions. 

(ii)  Watery  alcoholic  solutions. 
Watery  alcoholic  solutions  are  prepared  by  mixing 

Filtered  alcoholic  solution  of  the  dye,         -  1  to  5  c.c. 

Distilled  water,        -  100  c.c. 

Filter  immediately  before  use. 

These  solutions  are  seldom  used  in  this  form,  as  they  do  not  keep  well : 
it  is  simpler  to  make  them  up  as  required  by  pouring  several  cubic  centimetres 
of  water  into  a  porcelain  dish,  and  adding  to  it  a  few  drops  of  the  filtered 
alcoholic  solution  until  an  iridescent  pellicle  with  a  metallic  lustre  appears 
covering  the  surface. 

(iii)   Aqueous  solutions. 
Mix  in  a  small  bottle 

Dye,       -  0'25  gram. 

Distilled  water,  25        c.c. 

Shake  and  leave  the  water  standing  on  the  dye.     Filter  before  use. 


138  STAINED   PREPARATIONS 

The  above  proportions  give  a  saturated  solution  and  there  should  be  an 
excess  of  the  dye  at  the  bottom  of  the  bottle. 

These  solutions  are  very  little  used  :  they  do  not  keep  well  and  should 
be  prepared  as  required.  They  stain  slowly  but  sharply. 

Aqueous  solutions  of  quinoline  blue,  vesuvin,  methyl-green  and  neutral- 
red  are  used  for  staining  living  organisms. 

B.  Staining  solutions  containing  a  mordant. 

(i)  Carbolic  acid  solutions. 
These  are  more  used  than  any  other  stains  and  retain  their  properties 

for  a  very  long  time.  .  ,7,         ,   ,  .    ,    . 

Liehl  s  carooi-juchsin. 

Basic  fuchsin,  1  gram. 

Carbolic  acid  crystals,  -                                                                         5  grams. 

Absolute  alcohol,     -  10  c.c. 

Distilled  water,        -  -       100  „ 

Rub  up  the  fuchsin  and  alcohol  in  a  glass  mortar,  add  the  carbolic  acid  and 
mix  ;  add  two-thirds  of  the  water  little  by  little,  stirring  all  the  time  ;  pour 
the  mixture  into  a  bottle  then  rinse  out  the  mortar  with  the  remainder  of 
the  water  and  add  it  to  the  mixture  in  the  bottle.  Leave  for  24  hours  before 
filtering  into  a  clean  ground-glass  stoppered  bottle. 
A  diluted  solution  prepared  as  follows  is  often  used  : 

MJX  Dilute  carbol-fuchsin. 

Ziehl's  carbol-fuchsin,       -  1  c.c. 

Distilled  water,        -  3  to  10  c.c. 

Mix  and  filter  just  before  use. 

Carbol-gentian- violet  (Nicolle) . 

Gentian-violet.         -  1  gram. 

Carbolic  acid  crystals,  -  2  grams. 

Absolute  alcohol,     -  -         10  c.c. 

Distilled  water,        -  -       100     „ 

Prepare  as  in  the  case  of  carbol-fuchsin.  Use  as  such.  This  solution  is 
•chiefly  used  for  Gram's  stain. 

Carbol-crystal-violet  ( Roux) . 

Substitute  crystal-violet  for  gentian-violet  and  prepare  in  the  same  way 
as  the  preceding  solution. 

Crystal-violet  has  the  advantage  over  gentian-violet  of  being  a  well-defined 
crystalline  compound.  Gentian-violet  is  an  amorphous  product  which  varies  in 
composition.  Crystal- violet  however  is  not  so  powerful  a  dye  as  gentian- violet. 

Carbol-thionin  (Nicolle). 

Thionin,  0'5  to  1  gram 

Carbolic  acid  crystals,  -  1  gram 

90  per  cent,  alcohol,  -  -         10  c.c. 

Distilled  water,        -  -       100     „ 

Prepare  in  the  same  way  as  carbol-fuchsin.  This  stain  is  recommended  for 
sections  and  films  ;  it  stains  rather  more  slowly  but  gives  better  results  than 
crystal-violet  and  gentian-violet  and  does  not  overstain. 

Carbol-methylene-blue  (Kuhne). 


Methylene  blue, 
Carbolic  acid  crystals, 
Absolute  alcohol,     - 
Distilled  water, 

Prepare  in  the  same  way  as  the  foregoing  solutions. 


1*5  to  2  grams. 
2  grams. 
10  c.c. 

100     , 


STAINING  SOLUTIONS  139 

U una's  polychrome  Hue. 

Unna's  polychrome  blue  solution  (Griibler),  -                    -       100  c.c. 

Carbolic  acid  crystals,      -  1  gram. 

90  per  cent,  alcohol,          -  -         10  c.c. 

Distilled  water,        -  -         Q.S.  to  100  c.c. 

Dissolve  the  carbolic  acid  in  the  alcohol,  add  sufficient  water  to  make  up  to 
80  c.c.  and  then  add  the  polychrome  blue. 

(ii)  Aniline  solutions. 

These  solutions  keep  badly  and  should  be  freshly  prepared  every  time  they 
are  wanted.  They  have  no  advantage  over  carbolic  solutions  and  are  gradu- 
ally dropping  out  of  use. 

In  preparing  them,  the  following  solution  must  first  be  made  up  : 

Aniline  oil  water. 

Aniline  oil,      -  5  c.c. 

Distilled  water,        -  -       100     „ 

Mix  the  oil  and  water  in  a  yellow  glass  bottle,  shake  vigorously  and  leave 
them  in  contact.  Just  before  use  filter  the  solution  through  a  previously 
moistened  filter  paper.  See  that  no  fine  droplets  of  oil  pass  through  the  filter 
as  this  would  spoil  the  results  of  the  staining ;  should  this  accident  occur, 
filter  the  solution  again. 

Ehrlich's  aniline-violet. 

Filter  into  a  porcelain  dish  about  10  c.c.  of  aniline  oil  water.  To  the 
filtrate  add  a  few  drops  of  a  filtered  alcoholic  solution  of  gentian-violet  until 
an  iridescent  pellicle  appears.  Use  the  solution  at  once.  It  should  be  freshly 
prepared  every  day. 

Aniline-fuchsin,  aniline-crystal-violet  and  aniline-methylene-blue  are  all 
prepared  in  the  same  way. 

(iii)  Alkaline  solutions. 

These  solutions  have  been  extensively  used  in  Germany.  -  Almost  the  only 
alkaline  solution  now  used  however  is  Lceffler's  alkaline  methylene  blue. 
Sorrel's  blue  (vide  Hcematozoa)  is  an  alkaline  dye.  [Borax  blue  also  is  not 
infrequently  used  for  staining  some  of  the  hsematozoa  (q.v.).] 

Loeffler's  alkaline  methylene  blue. 

Alcoholic  solution  of  methylene  blue,  -         30  c.c. 

1  in  10,000  aqueous  solution  of  caustic  potash,  -  -       100    „ 

Mix  in  a  bottle  and  filter  before  use.  This  solution  is  rapidly  decomposed  by 
the  caustic  potash  combining  with  the  C02  of  the  atmosphere. 

Kuhne's  alkaline  blue. 

Alcoholic  solution  of  methylene  blue,  -         30  c.c. 

1  per  cent,  aqueous  solution  of  ammonium  carbonate,  -       100     ,, 

Mix  and  filter  before  use.     This  solution  keeps  better  than  Lceffler's. 

(iv)  Perchloride  solutions. 

Nastikow's  violet. 

1  in  2000  aqueous  solution  of  perchloride  of  mercury,  -         10  c.c. 

Alcohol  solution  of  gentian-violet,     -  1     „ 

Mix  and  filter.     This  stain  does  not  keep  well. 


140  STAINED   PREPARATIONS 

(v)  Complex  stains. 
Roux's  blue. 
SOLUTION  A. 

Violet  dahlia,  1  gram. 

Absolute  alcohol,     -  -         10  grams. 

Distilled  water,        -  -        Q.S.  for  100  grams. 

SOLUTION  B. 

Methyl-green,  2  grams. 

Absolute  alcohol,     -  20       „ 

Distilled  water,        -  -        Q.S.  for  200  grams. 

1.  Prepare  each  solution  separately  by  rubbing  up  the  dye  with  the  alcohol 
in  a  mortar  and  add  the  water  gradually.     Let  the  mixture  stand  for  24  hours 
in  a  bottle. 

2.  Then  mix  the  two  solutions,  filter  and  store  in  a  well-stoppered  bottle. 

2.  Simple  staining-. 

For  purposes  of  staining  there  should  be  at  hand — 

(a)  Several  small  glass  funnels  and  a  number  of  pieces  of  filter   paper 
folded  to  fit  them.     Staining  solutions  ought  always  to  be  filtered  before 
being  used  and  should  be  dropped  from  the  filter  straight 
on  to  the  preparation. 

(b)  A  wash-bottle  filled  with  water  recently  filtered  through 
a  Chamberland  filter  (fig.  126).     This  bottle  is  so  arranged 
that  by  simply  tilting  it  the  water  runs  out  through  the 
glass  tube. 

(c)  In  the  absence  of  a  sink,  a  large  glass  dish  to  collect 
the  washings. 

(d)  A  number  of  slides  and  cover-glasses,  a  pair  of  Cornet's 
or  Debrand's  forceps,  platinum  needles,  a  piece  of  soft  cloth, 
some  small  squares  of  filter  paper  or  a  packet  of  cigarette 
papers  and  a  few  Pasteur  pipettes. 

(e)  A  Bunsen  burner  with  a  pilot  flame. 

(i)  The  methods  of  staining  living  organisms. 

The  object  of  staining  living  organisms  is  to  make  them 
FlG'  1botS»W*dl"  more  readily  visible  for  microscopical  examination  while  at 
the  same  time  preserving  their  motility. 

For  this  purpose  aqueous  solutions  of  dyes  which  have  no  toxic  action  on 
the  organisms  are  used  e.g.  vesuvin  (Metchnikoff),  methyl-green  (Babes), 
quinoline  blue,  fuchsin,  neutral-red,  etc. 

Technique. — Make  the  preparation  in  the  same  way  as  for  the  examination 
of  unstained  living  organisms.  Invert  the  cover-glass  on  the  slide  and  run 
a  drop  of  a  watery  solution  of  the  dye  along  the  edge  of  the  cover-glass  ;  by 
capillary  action  it  will  be  drawn  between  the  cover-glass  and  slide. 

Or  if  preferred  a  small  drop  of  the  stain  can  with  a  very  fine  pipette  be 
added  to  the  culture  on  the  cover-glass  and  the  two  solutions  mixed  with  the 
end  of  the  pipette ;  the  cover-glass  is  then  inverted  on  the  slide  and  the 
preparation  is  ready  for  examination. 

(ii)  The  staining  of  dried  films. 

This  is  the  best  method  of  examining  the  morphology  of  micro-organisms 
and  it  gives  moreover  preparations  which  are  practically  permanent. 

Technique. — A.  1.  Pick  up  a  cover-glass  with  a  pair  of  Cornet's  forceps, 


SIMPLE   STAINING 


141 


place  a  drop  of  a  broth  culture  about  its  centre  and  spread  the  drop  with 
the  end  of  a  pipette  ;  or 

Place  a  small  drop  of  filtered  water  on  the  cover-glass,  mix  a  trace 
of  the  growth  from  a  solid  medium  with  it  and  spread  the  mixture  with  a 
platinum  wire. 

2.  Dry  the  film  gently  either  by  holding  it  above  the  pilot  flame  of  a 
Bunsen  or  by  placing  it  on  a  Koch's  drying  stage  (fig.  127)  heated  to  45° 
or  50°  C. 


FIG.  127. — Koch's  drying  stage. 

While  the  film  is  drying  keep  the  liquid  evenly  spread  over  the  cover-glass 
to  prevent  the  formation  of  concentric  circles. 

3.  NoWjfce  the  film  to  prevent  the  organisms  being  washed  off  by  the  stain, 
etc.     This  may  be  done  either  (a)  by  passing  the  cover-glass,  film  upwards, 
two  or  three  times  through  the  ordinary  Bunsen  flame  ;    the  organisms  are 
liable  to  be  distorted  and  shrivelled  by  this  procedure  so  that  it  is  better 
(b)  to  pour  two  or  three  drops  of  alcohol-ether  on  the  film  side  of  the  cover- 
glass  and  let  it  evaporate.     This  method  produces  no   distortion   of  the 
organisms. 

Alcohol-ether. 

Absolute  alcohol,     -  -         50  c.c. 

Ether  pur.,     -  50     „ 

In  special  cases  it  is  better  to  fix  the  films  by  immersing  them  in  absolute  alcohol 
for  15  or  20  minutes  or  by  exposing  them  to  the  vapour  of  osmic  acid  (vide  Tre- 
ponema  pallidum). 

4.  Filter  two  or  three  drops  of  stain  straight  on  to  the  film  (diluted  carbol- 
fuchsin,  carbol-thionin,  alkaline  blue,  etc.,  may  any  of  them  be  used).     Be 
careful  not  to  let  the  stain  run  on  to  the  under  surface  of  the  cover-glass. 
Stain  for  J  to  1  minute. 

5.  Wash  off  the  stain  by  running  a  gentle  stream  of  water  from  the  wash- 
bottle  on  to  a  corner  of  the  cover-glass.     The  water  ought  not  to  be  poured 
on  to  the  centre  of  the  film  for  fear  of  washing  it  off. 

6.  The  film  may  now  be  examined  (with  the  ^  in.  immersion  lens  and  a 
No.  II.  eyepiece,  for  preference)  : 

(a)  Provisionally,  in  water,  by  inverting  the  wet  cover-glass  on  to  a  slide, 
blotting  the  upper  surface  of  the  cover-glass  with  a  fine  cloth  [or  filter  paper] 
and  then  placing  a  drop  of  immersion  oil  on  the  blotted  surface. 

(b)  After  drying  and  mounting  in  Canada  balsam.     If  the  film  is  to  be 
mounted,  dry  the  cover-glass  either  in  the  air  or  by  heating  it  gently,  and 
place  a  small  drop  of  balsam  with  the  end  of  a  fine  glass  rod  on  the  film  side, 
invert  it  on  to  a  slide  and  press  gently  to  spread  the  balsam. 

To  sum  up  :  Spread  a  drop  of  culture  on  a  cover-glass,  dry,  fix,  stain,  wash 
in  water,  dry,  mount  in  balsam  and  examine. 

Notes. — (a)  It  is  important  in  staining  films  to  remember  which  is  the  film  side 
of  the  cover-glass.  If  this  should  be  forgotten,  gently  scratch  the  surfaces  of  the 


142  STAINED   PREPARATIONS 

cover-glass  near  the  edge  with  the  point  of  a  needle,  and  the  side  on  which  the  film 
has  been  spread  will  be  easily  distinguished  by  the  little  scratches  which  will  remain. 
But  the  possibility  of  losing  the  film  side  can  be  avoided  by  marking  the  upper 
limb  of  the  forceps  with  a  glass  pencil,  or  by  the  use  of  a  pair  of  Cornet's  forceps 
on  one  limb  of  which  a  small  knob-like  depression  is  impressed  in  the  metal ;  if 
the  forceps  be  always  used  with  this  knob  upwards  it  will  act  as  a  guide. 

(6)  A  small  quantity  only  of  culture  should  be  used  for  making  the  film.  The 
shape  of  the  organisms  can  best  be  made  out  when  there  are  only  a  few  in  the  field 
of  the  microscope. 

(c)  The  Canada  balsam  should  be  dissolved  with  xylol  and  the  solution  should  be 
of  such  a  consistency  that  it  does  not  tail  when  a  drop  is  taken  out  with  the  glass 
rod.     Balsam  should  be  kept  in  a  bottle  stoppered  with  a  glass  bell- stopper  and 
having  a  rim  arranged  so  that  the  excess  of  balsam  taken  up  on  the  glass  rod  can 
be  drained  off. 

(d)  Alcohol-ether,    alcohol    and    volatile    reagents    generally    are    best    kept    in 
drop-bottles  stoppered  with  ground-glass  stoppers  (several  patterns  can  be  obtained 
at  the  shops) :    shallow  thick  glass  bottles  of  60  to  100  c.c.  capacity  are  perhaps 
the  most  convenient. 

B.  The  method  which  has  just  been  described  is  especially  useful  for  delicate 
work  and  for  making  films  which  are  to  be  preserved.  But  for  the  provi- 
sional examination  of  cultures  and  for  routine  work  it  is  quicker  and  more 
economical  to  work  with  slides. 

1.  Take  a  slide  between  the  fingers  or  hold  it  in  a  pair  of  Debrand's  forceps, 
and  place  a  little  drop  of  the  culture  on  it. 

2.  Spread,  dry  and  fix  as  in  the  former  case  (A). 

3.  Stain  the  film  and  wash  it  in  the  manner  described,  and  then  dry  the 
slide.     Put  a  drop  of  cedar- wood  oil  straight  on  to  tlie  film  without  using 
a  cover-glass  and  examine  with  the  oil-immersion  lens. 

If  after  examination  it  is  desired  to  preserve  the  preparation  the  cedar- wood  oil 
can  be  washed  off  with  a  few  drops  of  xylol,  and  when  this  has  evaporated  the 
slide  is  put  away  dry.  Or  after  washing  off  the  cedar  oil  the  film  may  be  mounted 
with  a  drop  of  balsam  and  cover-glass. 

[C.  Another  method  which  gives  excellent  results  and  is  commonly  adopted 
by  us  seems  to  deserve  description  here. 

[1.  The  film  is  spread,  dried  and  fixed  on  a  slide  as  in  B. 

[2.  Wash  the  film  for  a  moment  or  two  in  a  10  per  cent,  aqueous  solution 
of  acetic  acid.  Wash  thoroughly  in  water.  Blot  and  dry. 

[3.  Place  a  drop  of  the  stain  on  the  centre  of  the  film  and  lower  a  cover- 
glass  on  to  the  stain,  avoiding  the  introduction  of  air  bubbles.  Blot  the 
upper  surface  of  the  cover-glass  with  blotting  paper. 

[4.  Put  a  drop  of  oil  on  the  dried  surface  of  the  cover-glass  and  examine 
with  a  y^th  oil  immersion. 

[If  the  preparation  is  to  be  preserved,  float  off  the  cover-glass  from  the 
slide  by  putting  a  drop  or  two  of  water  at  the  edge  of  the  cover-glass.  Wash 
the  slide  in  water,  blot  and  dry  it.  The  slide  may  then  be  kept  indefinitely. 

[Both  in  this  method  and  in  the  preceding,  the  film  may  be  decolourized 
in  a  10  per  cent,  aqueous  solution  of  acetic  acid  or  in  alcohol,  and  then  restained 
with  another  dye  ;  so  that  using  the  same  film  one  may  first  determine  the 
morphology  of  the  organism  by  examination  in  a  simple  stain  and  then 
ascertain  its  reaction  to  Gram's  stain  (vide  infra).] 

3.  Grain's  method  of  staining. 

Gram  devised  a  method  of  staining  which  serves  to  divide  bacteria  into  two 
large  groups. 

Some  bacteria,  when  stained  with  a  basic  pararosanilin  dye  in  aniline  or 


GRAM'S   STAIN  143 

carbolic  solution  and  treated  afterwards  with  a  special  mordant  containing 
iodine,  are  not  decolourized  by  absolute  alcohol  and  similar  decolourizing 
agents.  The  anthrax  bacillus  is  an  example  of  this  group. 

On  the  other  hand,  other  bacteria  when  treated  in  the  same  way  are  readily 
decolourized  with  absolute  alcohol,  e.g.  the  typhoid  bacillus. 

Bacteria  then  are  classified  with  reference  to  these  reactions  into  two 
groups,  termed  gram-positive  (organisms  which  retain  the  stain)  and  gram- 
negative  (organisms  which  are  decolourized).  The  anthrax  bacillus  is  said 
to  be  gram-positive,  and  the  typhoid  bacillus  gram-negative. 

The  mordant  has  the  following  composition  : 

Gram's  (or  Lugol's)  solution. 

Iodine,  -  1  gram. 

Potassium  iodide,    -  2  grams. 

Distilled  water,        -  300  c.c. 

In  the  original  method  absolute  alcohol  was  used  as  the  decolourizing  agent. 
But  pure  aniline  oil  (Weigert)  or  acetone  alcohol  (Nicolle)  are  now  sometimes 
used  in  its  place. 

Acetone  alcohol. 

Absolute  alcohol,     -  5  parts. 

Acetone,          -  1  part. 

According  to  Nicolle,  a  bromine- bromide,  iodine- bromide,  or  bromine- iodide 
solution  may  any  of  them  be  used  in  place  of  Gram's  solution.  They  are  all 
prepared  in  the  same  proportions  as  Gram's  iodine-iodide  solution. 

Gram's  stain  has  undergone  many  modifications,  and  is  used  as  a  double 
stain  for  films,  sections,  etc.  These  modifications  will  be  dealt  with  in  a 
special  chapter  and  for  the  present  the  use  of  this  classical  method  as  a  means 
of  diagnosis  will  alone  be  considered. 

Technique. — 1.  Prepare  a  film  on  a  slide  or  cover-glass. 

2.  Stain  for  30  to  60  seconds  with  carbol-gentian-violet. 

3.  Blot  up  the  excess  of  stain  (but  do  not  wash),  drop  two  or  three  large 
drops  of  Gram's  solution  on  the  film  and  let  it  act  for  20  to  30  seconds.     The 
preparation  will  have  now  assumed  a  brown  tint. 

4.  Wash  in  water  and  dry. 

5.  Pour  absolute  alcohol  over  the  film  a  drop  at  a  time  until  no  more 
violet  stain  comes  away — usually  20  to  30  seconds  (Notes  (a)  and  (b)  infra). 

6.  Wash  in  water  quickly. 

7.  Examine  the  film  in  water.     If  the  organisms  are  gram-positive  they 
are  stained  deep  violet,  but  if  gram-negative  decolourized :  sometimes  some 
of  the  organisms  will  be  decolourized  while  others  are  still  stained  violet ; 
in  that  case  a  further  washing  in  alcohol  will  complete  the  reaction. 

[Many  bacteriologists  prefer  to  counterstain  the  film.  For  this  purpose, 
after  washing  in  water  (Stage  6)  the  film  is  flooded  with  some  weak  staining 
solution  the  colour  of  which  is  in  sharp  contrast  with  violet.  Dilute  carbol- 
fuchsin  (1-5  or  1-10)  or  bismarck  brown  (p.  136)  is  convenient ;  the  former 
is  allowed  to  act  for  about  J  minute,  while  bismarck  brown  requires  rather 
longer  (2  minutes).  Wash  in  water,  blot  and  dry.  Gram-positive  organisms 
are  as  in  the  former  case  stained  violet,  while  gram-negative  organisms  being 
decolourized  by  the  alcohol  take  the  counterstain  and  appear  pink  or 
brown  as  the  case  may  be.] 

To  keep  the  cover-glass  preparation,  dry  and  mount  in  balsam.  If  the 
film  was  made  on  a  slide  it  merely  requires  to  be  dried. 

To  sum  up  :  Prepare  and  fix  a  film,  stain,  treat  with  iodine  solution,  wash> 
dry,  treat  with  alcohol,  wash  [counterstain,  wash,]  dry  and  examine. 


144  STAINED   PREPARATIONS 

Notes. — (a)  Stage  5,  decolourization,  is  a  delicate  manipulation.  The  length  of 
time  during  which  decolourization  must  be  continued  varies  with  the  intensity  of 
the  stain  used,  the  length  of  time  during  which  it  is  allowed  to  act,  the  number 
of  organisms,  etc. ;  practice  and  a  certain  amount  of  skill  are  more  than  any  rules 
the  secrets  of  success.  It  is  obvious  that  insufficient  decolourization  of  a  gram- 
negative  organism  may  lead  to  mistakes ;  on  the  other  hand  the  most  resistant 
bacteria  can  be  decolourized  by  prolonging  unduly  the  action  of  the  alcohol,  and 
such  treatment  might  result  in  a  gram-positive  organism  being  classed  with  the 
gram-negative  group. 

[(6)  In  view  of  the  difficulty  as  to  the  time  of  decolourization  we  have  found 
it  useful,  especially  for  beginners  and  in  dealing  with  organisms  such  as  the 
meningococcus,  to  prepare  on  the  same  slide  three  separate  films.  At  one  end  there 
will  be  a  film  of  a  gram-positive  organism  e.g.  Staphylococcus,  at  the  other  end  a 
gram-negative  organism  e.g.  Bacillus  coli  communis,  and  in  the  centre  the  organism 
whose  reaction  is  to  be  tested  e.g.  Meningococcus.  The  films  are  stained  and 
decolourized  as  described  above,  and  then  examined  in  water.  If  decolourization  is 
sufficient,  the  staphylococci  are  all  violet  and  the  colon  bacilli  all  pink  (or  brown). 
If  the  films  have  been  under- decolourized  some  of  the  bacilli  will  be  stained  violet, 
and  if  over- decolourized  some  of  the  cocci  will  be  pink  (or  brown).  When  it  is 
evident  that  the  decolourization  has  been  correctly  done  the  organism  whose 
reaction  is  being  tested  is  examined.] 

(c)  Films  prepared  by  Gram's  method  do  not  keep  so  well  as  when  stained  with 
ordinary  stains  and  ultimately  become  decolourized. 

4.   Claudius'  method. 

Claudius  suggested  a  method  of  staining  which,  while  having  all  the 
advantages,  has  both  a  simpler  technique  and  gives  more  constant  results 
than  Gram's  method.  Thus  the  bacillus  of  malignant  oedema  and  the 
bacillus  of  quarter  ill  are  somewhat  readily  decolourized  by  Gram's  method 
but  retain  the  stain  well  by  Claudius'  method. 

The  author  repeated  Claudius'  experiments  and  obtained  results  which  fully 
confirm  that  observer's.  Claudius'  method  has  many  advantages  for  the  student ; 
beginners  using  Gram's  method  never  know  when  to  stop  decolourizing,  sometimes 
they  leave  the  alcohol  on  too  long  and  sometimes  they  do  not  let  it  act  for  long 
enough,  and  in  either  case  the  results  are  unsatisfactory.  These  difficulties  do  not 
arise  in  Claudius'  method. 

The  following  solutions  are  required  : 

(a)  A  1  per  cent,  aqueous,  solution  of  methyl  violet  6B  (or  a  solution  of 
carbol-gentian  violet). 

(b)  A  solution  of  picric  acid. 

Saturated  solution  of  picric  acid,        -  1  volume. 

Distilled  water,        -  1       „ 

Technique. — 1.  Prepare  and  fix  a  film  in  the  ordinary  way. 

2.  Stain  with  violet  for  1  minute. 

3.  Wash  in  water,  and  blot  up  the  excess. 

4.  Treat  with  the  picric  acid  solution  for  1  minute  and  blot. 

5.  Decolourize  with  chloroform  or  clove  oil  until  the  decolourizing  agent  is 
no  longer  tinted  blue. 

6.  Examine  in  clove  oil  or  mount  in  balsam. 


CHAPTER  IX. 

THE  STAINING  OF  SPORES,  CAPSULES  AND  FLAGELLA. 
THE  STUDY  OF  THE  MOTILITY  OF  BACTERIA. 

Section  I. — Spores,  p.  145. 

1.  The  examination  of  unstained  preparations,  p.  145.     2.  The  staining  of  spores, 
p.  146. 

Section  II. — The  staining  of  capsules,  p.  147. 
Section  III. — The  staining  of  flagella,  p.  148. 

1.  The  staining  of  flagella  in  living  organisms,  p.  148.     2.  The  staining  of  flagella 
in  dried  preparations,  p.  149. 
Section  IV. — The  methods  of  studying  the  motility  of  micro-organisms,  p.  154. 


SECTION  I.— SPORES. 

WITHIN  the  protoplasm  of  certain  micro-organisms,  a  small  bright  refrac- 
tile  spot  is  seen  at  one  period  or  another  of  their  existence.  This  refractile 
body,  which  does  not  stain  readily  with  the  ordinary  aniline  dyes,  is  known 
as  a  spore,  or  more  strictly  an  endospore.  The  occurrence  of  spores  was 
first  described  by  Pasteur. 

On  the  death  or  destruction  of  a  spore- bearing  organism  the  spores  are  set  free 
from  the  protoplasm  in  which  they  originated.  They  are  surrounded  by  a  highly 
resistant  membrane,  which  not  only  renders  them  immune  to  the  agents  ordinarily 
destructive  of  bacteria,  but  also  prevents  them  becoming  stained  by  the  methods 
generally  employed  for  staining  micro-organisms. 

Endospore  formation  does  not  occur  in  all  bacteria  :  it  is  unknown  in  the 
micrococci,  in  which  the  resistant  form  is  due  to  a  thickening  of  the  enveloping 
membrane  and  is  known  as  an  arthrospore.  Arthrospores  differ  from  endo- 
spores  in  that  they  react  to  stains  in  the  same  way  as  do  their  corresponding 
organisms. 

It  is  therefore  only  necessary  to  describe  the  methods  of  staining  endospores. 
The  organisms  more  commonly  used  for  illustrating  the  methods  are  the 
Bacillus  anthracis,  Bacillus  megatherium,  Bacillus  maligni  oedematis,  Bacillus 
tetani,  and  Bacillus  subtilis. 

1.  Examination  of  unstained  preparations. 

In  unstained  preparations  the  spore  appears  as  a  small,  refractile,  spherical 
or  oval  spot  within  the  protoplasm  of  the  cell ;  it  is  surrounded  by  a  bright 
refractile  ring,  and  is  always  smaller  than  the  mother  cell.  The  mother 
cell  gives  rise  to  a  single  spore  which  becomes  free  on  the  disappearance  of 
the  cellular  protoplasm  ;  the  spore  in  turn  germinates,  giving  origin  to  a 
new  bacterium. 


146  SPORE   STAINING 

All  these  facts  can  be  observed  under  the  microscope  in  a  hanging  drop 
culture  of  the  anthrax  bacillus  (p.  134).  When  it  is  desired  merely  to  deter- 
mine the  presence  of  spores  in  bacteria  an  ordinary  film  is  made  on  a  slide 
as  described  on  pp.  140  and  141. 

2.  The  staining  of  spores. 

When  spore-bearing  organisms  are  stained  with  the  basic  aniline  dyes  the 
spores  do  not  take  up  the  dye  and  appear  as  unstained  spots  in  the  stained 
bacilli.  To  stain  the  spore  it  is  necessary  therefore  to  apply  special  methods 
which  have  been  designed  to  overcome  its  resistance  to  staining  reagents. 

(i)   Simple  staining. 

This  method  stains  both  the  bacilli  and  the  spores. 

A.  Method  recommended. — 1.  Prepare  a  cover-glass  film  of  the  culture  to 
be  examined  and  dry  it. 

2.  Pass  the  cover-glass,  film  side  uppermost,  ten  times  through  the  heating 
flame  of  the  Bunsen,  but  sufficiently  quickly  to  prevent  the  preparation  being 
scorched. 

3.  Stain  with  carbol-violet  for  15  to  30  minutes. 

4.  Wash,  dry,  mount  in  balsam.     Examine.     The  bacteria  and  the  spores 
are  both  stained  violet. 

B.  Chromic  acid  method. — 1.  Make  a  film  on  a  cover-glass  and  dry  it. 

2.  Drop  a  large  drop  of  a  1  in  20  aqueous  solution  of  chromic  acid  on  the 
film,  and  leave  it  for  4  or  5  minutes. 

3.  Wash  in  water. 

4.  Stain  in  carbol-violet  for  15  minutes  to  half  an  hour. 

5.  Wash.     Mount.     Examine. 

(ii)  Double  staining. 

The  object  of  double  staining  is  to  differentiate  the  spore  from  the  bacillus 
by  staining  the  bacillus  one  colour  and  the  spore  a  different  colour. 

Principle  of  the  method. — Spores  stain  with  difficulty,  but  once  stained 
they  retain  the  dye  with  more  tenacity  than  the  bacillary  protoplasm,  so 
that  decolourizing  agents  will  decolourize  the  latter  before  they  take  the 
stain  out  of  the  spores. 

A.  Method  recommended. — 1.  Prepare  a  cover-glass  film,  dry  and  fix  it  by 
passing  it  rapidly  through  the  flame  two  or  three  times. 

2.  Drop  a  large  drop  of  carbol-fuchsin  on  the  film  and  warm  it  over  a 
small  flame  until  steam  just  begins  to  rise,  then  keep  the  solution  warm  for 
4  or  5  minutes  by  moving  it  about  over  the  flame.1     Both  the  bacilli  and 
the  spores  are  now  stained  an  intense  red. 

3.  Wash  in  water. 

4.  Decolourize  for  a  few  seconds  in  a  solution  of  nitric  acid  : 

Pure  nitric  acid,      -  1  part. 

Distilled  water,        -  3  parts. 

The  bacilli  should  be  decolourized  while  the  spores  are  still  stained  red. 

5.  Wash  well  in  water. 

6.  Counterstain  with  a  drop  of  diluted  alcoholic  solution  of  methylene 
blue  for  30  to  60  seconds.     The  decolourized  bacilli  take  up  the  blue  stain. 

7.  Wash.     Dry.     Mount  in  balsam. 

The  bacilli  are  stained  blue  ;    the  spores  red. 

[x  This  may  be  done  on  a  warm  stage  (p.  141,  fig.  127)  taking  care  to  select  a  place  where 
the  metal  is  not  too  hot.] 


SPORE   STAINING  147 

Note. — This  method  gives  excellent  results  with  B.  megatherium  but  is  not  so 
good  for  B.  anthracis  :  absolute  alcohol  is  a  better  decolourizing  agent  for  the  latter. 
Decolourization  is  in  fact  the  difficult  part  of  double  staining,  but  after  a  few 
trials  the  extent  to  which  decolourization  must  be  pushed  to  decolourize  the  bacilli 
while  leaving  the  spores  stained  can  be  determined  for  different  organisms. 

B.  Mceller's  method. — 1.  Make  a  cover-glass  film.     Dry.     Fix  in  absolute 
alcohol  for  2  minutes,  then  in  chloroform  for  2  minutes.     Dry. 

2.  Drop  a  few  drops  of  a  1  in  20  aqueous  solution  of  chromic  acid  on  the 
film  and  leave  for  4  or  5  minutes.      Wash  in  water. 

3.  Stain  in  carbol-fuchsin  in  the  warm  as  described  above  (A).     Wash, 
in  water. 

4.  Decolourize  for  a  few  seconds  in  a  5  per  cent,  solution  of  sulphuric  acid 
and  complete  the  decolourization  in  absolute  alcohol. 

5.  6,  7.     Wash.     Stain  in  blue.     Mount. 

C.  Aladar-Aujeszky's    method. — 1.  Make    a    cover-glass    film.     Dry    in 
the  air. 

2.  Dip  the  preparation  for  2  to  4  minutes  into  a  porcelain  capsule  con- 
taining a  0'5  per  cent,  solution  of  pure  hydrochloric  acid  which  has  been 
heated  but  not  boiled. 

3.  Wash  freely  in  water.     Dry.     Fix  in  the  flame. 

4.  Stain  with  carbol-fuchsin  in  the  warm  by  heating  until  steam  rises  : 
as  the  stain  evaporates  add  a  fresh  supply. 

5.  Decolourize  rapidly  in  a  4  per  cent,  solution  of  sulphuric  acid. 

6.  Wash.     Stain  in  blue.     Mount. 

D.  Orszag's  method. — 1.  Place  a  small  drop  of  the  following  mixture  on 
a  cover-glass. 

0'5  per  cent,  aqueous  solution  of  sodium  sajicylate,     -         -          4  parts. 
5  per  cent,  aqueous  solution  of  acetic  acid,  1  part. 

Make  an  emulsion  of  the  organisms  in  this  solution.     Dry.     Fix  the  film  in 
the  flame. 

2.  Stain  with  carbol-fuchsin  in  the  warm  for  2  minutes. 

3.  Decolourize  with  a  1  per  cent,  aqueous  solution  of  sulphuric  acid. 

4.  Wash.     Counterstain  with  blue.     Mount  as  before. 

E.  Thesing's  method. — 1.  Prepare  a  cover-glass  film.     Dry.     Fix  in  the 
flame. 

2.  Place  a  drop  of  a  1  per  cent,  aqueous  solution  of  platinum  chloride  on 
the  film.     Heat  to  boiling. 

3.  Wash  in  a  large  quantity  of  water.     Dry. 

4.  Stain  with  carbol-fuchsin  as  in  the  foregoing  methods. 

5.  Decolourize  with  33  per  cent,  alcohol. 

6.  Wash.     Dry.     Counterstain  with  blue.     Mount. 


SECTION  II.— THE   STAINING  OF  CAPSULES. 

Some  micro-organisms  are  surrounded  by  a  bright  hyaline  area  called  a 
capsule  which  can  be  demonstrated  by  certain  staining  devices.  When  these 
are  employed  the  organism  is  deeply  stained,  while  the  capsule  surrounding 
it  is  pale  with  a  feebly  stained  margin. 

A.  1.  Having  dried  and  fixed  a  film,  stain  for  1  minute  in  carbol-fuchsin. 
2.  Wash.     Treat  for  20-30  seconds  with  water  containing  1   per  cent, 
acetic  acid. 


148  CAPSULE   STAINING 

3.  Wash.     Dry.     Mount  in  balsam. 

In  the  author's  hands  this  method  has  given  better  results  than  the  following. 

The  method  may  be  modified  by  treating  the  film  first  with  a  1  per  cent,  solution 
of  acetic  acid  for  1  minute,  then  drying  it  and  afterwards  staining  with  carbol- 
violet. 

Simple  staining  with  dilute  carbol-fuchsin  also  gives  quite  good  results. 


B.  1.  Dry  and  fix  a  film  on  a  cover-^ 

2.  Stain  with  a  drop  of  the  following  solution  for  30-60  seconds  : 

Acetic  violet. 

Acetic  acid,    -  1  gram. 

Alcoholic  solution  of  gentian -violet,  or  crystal- violet,  -  5  c.c. 

Distilled  water,        -  100  grams. 

3.  Wash.     Dry.     Mount  in  balsam. 

C.  Rsebiger  suggests  staining  the  dried  but  unfixed  films  in  the  following 
solution,  which  must  be  filtered  : 

Gentian-violet,         -  -         15  grams. 

Commercial  formalin,       -  -       100       ,. 

After  staining,  wash,  dry  and  mount  in  balsam.     The  bacteria  are  stained 
violet  and  the  capsules  violet  with  a  pink  tint. 

D.  Nicolle  recommends  staining  with  carbol-gentian-violet  followed  by 
rapid  decolourization  in  a  1  in  3  solution  of  acetone-alcohol.     Mount  and 
examine  in  water. 

Hiss  fixes  the  films  in  the  flame,  stains  in  a  5  per  cent,  aqueous  solution  of  gentian- 
violet  or  fuchsin  in  the  warm,  then  washes  in  a  20  per  cent,  solution  of  copper 
sulphate,  dries  and  mounts  in  balsam. 

He  also  recommends  staining  in  a  half -saturated  aqueous  solution  of  gentian- 
violet  followed  by  washing  in  a  0*25. per  cent,  aqueous  solution  of  potassium  car- 
bonate. The  films  should  be  examined  in  a  drop  of  the  potassium  carbonate 
solution. 

The  staining  of  capsules  in  sections  requires  special  methods  which  will  be 
studied  later  (vide  Pneumococcus}. 


SECTION  III.— THE  STAINING   OF  FLAGELLA. 

Flagella  are  the  organs  of  locomotion  of  the  motile  bacteria  and  are  only 
visible  in  the  living  unstained  condition  in  such  large  organisms  as  the  sulpho- 
bacteria  (Bacterium  photometricum,  Beggiatoa  roseopersinica,  etc.).  To 
demonstrate  flagella  in  other  motile  organisms  complicated  staining  methods 
have  to  be  adopted. 

1.  The  staining  of  flagella  in  living1  organisms. 
Straus'  method. 

1.  Place  a  drop  of  a  broth  culture  of  the  organism  on  a  slide. 

2.  Add  a  drop  of  carbol-fuchsin  diluted  with  three  or  four  parts  of  water 
and  mix  the  culture  with  the  stain. 

3.  Cover  with  a  cover-glass  and  examine  at  once  with  an  oil-immersion 
lens. 

The  bacilli  are  stained  an  intense  red  and  the  flagella,  which  will  be  seen 
especially  well  on  the  living  actively-motile  bacilli,  assume  a  pale  pink  colour 
with  deeper  red  points  scattered  along  their  length. 

Note. — This  is  a  very  rapid  method  but  it  only  succeeds  with  certain  organisms, 


FLAGELLUM  STAINING  149 

and  its  action  on  these  is  uncertain.  The  best  results  are  obtained  with  Vibrio 
cholerce  asiaticce,  V.  finkler-prior,  V.  metchnikowi.  The  method  fails  altogether 
to  stain  the  flagella  of  many  organisms 'such  as  B.  febris  entericce,  B.  coli  communis, 
B.  subtilis,  etc. 

2.  The  staining  of  flagella  in  dried  preparations. 
General  rules. 

1.  Take  a  small  quantity  of  a  young  agar  culture  and  make  a  perfectly 
homogeneous,  very  faintly  opalescent  emulsion  in  a  watch-glass  containing 
ordinary  water,  or  preferably  distilled  water. 

2.  Fix  an  absolutely  clean  cover-glass  in  a  pair  of  Cornet's  forceps,  pass  it 
through  the  flame,  and  when  cool  place  a  drop  of  the  emulsion  on  it  with  a 
pipette.     Unless  the  cover-glass  be  perfectly  clean  the  liquid  will  not  spread 
uniformly. 

3.  By  tilting  the  cover-glass  run  the  liquid  over  the  surface,  then  let  the 
excess  gravitate  to  one  corner  and  aspirate  it  with  a  pipette. 

4.  Dry  the  film  in  the  air  away  from  dust  and,  without  fixing,  stain  by  one 
of  the  methods  described  below. 

By  following  the  above  instructions,  a  dilution  is  obtained  such  that  each  field 
of  the  microscope  contains  only  a  few  organisms,  which  is  an  essential  condition 
for  good  results.  By  this  method,  also,  the  mucoid  substances  which  agglomerate 
organisms  in  cultures  and  form  precipitates  on  the  cover-glass  (thus  obscuring  the 
details)  are  as  far  as  possible  excluded. 

(i)  Van  Ermengem's  method. 
(Method  recommended.) 

This  method  is  based  on  the  reduction  of  nitrate  of  silver  in  the  flagella  and 
gives  very  beautiful  preparations.  It  is  generally  used  in  preference  to  any 
other  method.  [Vide  also  method  (xi)  p.  153.] 

1.  Place  the  film  for  1  minute  at  50°  C.  (or  30  minutes  at  room  temperature) 
in  the  following  bath  which  must  be  freshly  prepared : 

2  per  cent,  aqueous  solution  of  osmic  acid,  8  c.c. 

10  per  cent,  aqueous  solution  of  tannin,     -  -         16     „ 

Glacial  acetic  acid,  1  drop. 

2.  Wash  in  water,  then  in  absolute  alcohol. 

3.  Treat  the  film  with  silver  for  1  or  2  minutes. 

Crystals  of  silver  nitrate,  1  gram. 

Distilled  water,        -  -      200  c.c. 

4.  Without   washing,   transfer   the   film   for   1    minute   to   the   reducing 

solution. 

Gallic  acid,     -  5  grams. 

Tannin,  3      „ 

Fused  sodium  acetate,  -  10      „ 

Distilled  water,        -  -      350  c.c. 

5.  Without  washing,  put  the  preparation  into  the  silver  bath  again  and 
keep  the  liquid  moving  over  the  film  until  the  latter  assumes  a  black  tint. 

6.  Wash.     Dry.     Mount  in  balsam. 

(ii)    Lceffler's  method. 

The  method  devised  by  Lceffler,  for  a  long  time  the  classical  method  of 
staining  flagella,  requires  a  good  deal  of  practice  and  gives  only  mediocre 
results.  The  films  are  often  covered  with  an  abundant  precipitate  which 
obscures  the  flagella  and  renders  their  detection  difficult. 


150  FLAGELLUM  STAINING 

The  following  reagents  are  required  : 

Fuchsin  ^  ink. 

25  per  cent,  aqueous  solution  of  tannin,     -  -         10  c.c. 

Cold  saturated  solution  of  ferrous  sulphate,  -                              5     „ 

Saturated  alcoholic  solution  of  fuchsin,       -  *                     1     „ 

Alkaline  solution. 

Alcoholic  soda,        -  1  gram. 

Distilled  water,        -  -       100  c.c. 

Acid  solution. 

Pure  sulphuric  acid,  1  gram. 

Distilled  water,        -  100  c.c. 

Staining  solution. 

Aniline  water,  -       100  c.c. 

1  per  cent,  solution  of  soda,      -  1     „ 

Gentian-violet  or  fuchsin,  4  to  5  grams. 

Shake.     Leave  for  a  few  hours  in  a  bottle.     Filter. 

Technique. — 1.  Mordanting.  Drop  on  to  a  film  prepared  as  above  (see  general 
rules)  a  large  drop  of  the  fuchsin  ink  containing  a  few  drops  of  the  acid  or  alkaline 
solution.  The  amount  of  the  latter  depends  upon  the  species  of  organism  under 
examination.  Heat  the  film  over  the  pilot  flame  of  a  Bunsen  until  steam  just 
begins  to  rise  and  continue  the  heating  for  30-50  seconds  :  be  careful  not  to  boil 
the  liquid.  This  stage  of  the  method  is  very  tricky  and  is  liable  to  failure. 

The  amount  of  the  acid  or  alkaline  solution  to  be  added  to  16  c.c.  of  fuchsin  ink 
has  been  determined  by  experiment.  The  following  table  shows  the  amounts 
necessary  for  the  principal  ciliated  bacteria. 

Mordant  alone  without  acid  or  alkali,  -  -       Spirillum  concentricum. 

Mordant  +  £-1  drop  of  acid  solution,      -  -       V.  cholerse  asiaticse. 

„       +6      drops          „         „  -       B.  pyocyaneus. 

„       + 18-20  „  „         „  -      Micrococcus  agilis. 

„       +20        „  „         „  -       B.  chauvsei. 

„       +  20-30  drops  of  alkaline  solution,    -  -       B.  febris  entericae. 

+  28-30      „  „  „  -       B.  subtilis. 

„       +26-28      ,,  „  ,,  -       B.  maligni  OBdematis. 

.    (from  20  drops  of  acid  solution        "\  T>     .„        f , ,          .,, 

"       +  {     to  15  drops  of  alkaline  solution, } 

2.  Washing. — Wash  in  water,  then  in  absolute  alcohol. 

3.  Staining. — Place  a  drop  of  the  staining  solution  on  the  film,  heat  until  steam 
begins  to  rise  gently,  and  let  the  hot  stain  act  for  about  a  minute. 

4.  Mounting. — Wash  in  a  large  volume  of  water  and    examine   in   water.     If 
satisfactory,  dry  and  mount  in  balsam. 

(iii)   Remy  and  Sugg's  method. 

This  method,  which  is  a  modification  of  Loeffler's,  is  designed  to  avoid  the 
formation  of  granular  precipitates.  The  fuchsin  solution  is  used  cold,  and 
after  staining  the  film  is  treated  with  iodine. 

Instead  of  Loeffler's  the  following  stain  is  used  : 

Staining  solution. 

Phenylamine  water,1       .-  -        20  c.c. 

Alcoholic  solution  of  gentian-violet,  -  1  drop. 

Distilled  water,        ...  5  c.c. 

Mix  the  gentian-violet  with  the  water  and  then  add  the  phenylamine  water. 

Technique. — 1.  The  mordanting  process  is  the  same  as  in  Lceffler's  method  but 
the  solution  is  left  on  the  film  for  15-30  minutes  and  is  not  heated. 

2.  Pour  off  the  mordant  and  replace  it  at  once  with  a  drop  of  Gram's  solution. 

3.  Wash  in  water,  then  in  absolute  alcohol. 

4.  Place  the  film  in  a  watch-glass  filled  with  the  stain  and  leave  it  for  half  an 
hour,  preferably  in  the  warm  (37°C.)  incubator. 

5.  Wash  in  water.     Examine  in  water.     Dry.     Mount  in  balsam. 

1  Prepare  in  a  similar  manner  to  aniline  oil  water  (p.  139). 


FLAGELLUM  STAINING  151 

(iv)   Nicolle's  and  Morax's  method. 
(Method  recommended.) 

This  method,  a  simplification  of  Loeffler's,  does  away  with  the  use  of  the 
acid  and  alkaline  solutions  and  is  a  satisfactory  stain  for  the  flagella  of  all 
motile  organisms.  Carbol-fuchsin  is  used  instead  of  Loeffler's  stain. 

1.  Mordanting. — Place  a  large  drop  of  fuchsin  ink  (without  any  addition 
of  acid  or  alkali)  on  the  film.     Heat  for  10  seconds  over  the  pilot  flame  of  a 
Bunsen. 

When  steam  begins  to  rise,  pour  off  the  solution,  tilt  the  cover-glass  and 
run  a  gentle  stream  of  water  from  a  wash-bottle  on  to  its  upper  angle  so  as 
to  wash  the  film  well  without  washing  the  organisms  away. 

Repeat  the  mordanting  and  washing  two  or  three  times.  Wipe  the  under 
surface  of  the  cover-glass  and  the  teeth  of  the  forceps  each  time  after 
washing :  otherwise  when  the  mordant  is  poured  on  again  the  solution  will 
run  under  the  cover-glass  and  along  the  forceps. 

2.  Staining. — Put  a  drop  of  carbol-fuchsin  on  the  film  and  heat  once  or 
twice  until  steam  has  been  rising  for  15  seconds. 

3.  Mounting. — Wash  and  examine  in  water.     If  the  preparation  be  satis- 
factory, dry  and  mount  in  balsam. 

Bunge's  and  de  Rossi's  methods. — These  methods  differ  slightly  from  that  of 
Nicolle  and  Morax  but  have  no  advantage  over  the  latter. 

Bunge's  mordant. 

Saturated  aqueous  solution  of  tannin,         -  3  parts. 

1  in  20  aqueous  solution  of  perchloride  of  iron,   -  1  part. 

To  ten  parts  of  this  mixture  add  one  part  of  a  saturated  aqueous  solution  of 
fuchsin.  The  mordant  must  be  exposed  to  the  air  for  a  few  weeks  before  use  ; 
while  so  exposed  it  acquires  a  brownish -red  colour. 

Filter  a  few  drops  of  the  above  solution  on  to  the  film  and  leave  it  for  5  minutes  : 
wash  in  water  and  dry :  stain  with  carbol-fuchsin  as  in  Nicolle  and  Morax's 
method  :  wash,  dry  and  mount  inbalsam. 

De  Rossi  treats  the  film  for  10  minutes  with  the  following  solution : 

Tannin,  5  grams. 

O'l  per  cent,  aqueous  solution  of  potash,    -  -       100  c.c. 

Wash   in   water.     Dry.     Stain  with   carbol-fuchsin  as  in  Nicolle's  and  Morax's 
method.     Wash.     Dry.     Mount. 

(v)   Trenkmann's  method. 

This  method  gives  satisfactory  results  but  it  takes  too  long  to  be  of  use 
in  routine  work. 

1.  Leave  the  film  in  the  following  solution  for  6-8  hours : 

Tannin,  2  grams. 

Distilled  water,        -  -          -       100  c.c. 

Pure  hydrochloric  acid,    -  4  drops. 

2.  Wash  in   water.     Treat  the  film  for   1    hour  in  a  watch-glass  containing  a 
saturated  solution  of  metallic  iodine  in  distilled  water. 

3.  Wash  in  water.     Stain  in  aniline-gentian-violet  for  half  an  hour. 

4.  Wash  in  water.     Examine.     Dry.     Mount  in  balsam. 

(vi)   Cerrito's  method. 

This  method  requires  a  good  deal  of  care,  because  the  mordant  frequently 
gives  rise  to  troublesome  deposits. 

The  mordant  consists  of  the  following  somewhat  complex  mixture : 
25  per  cent,  aqueous  solution  of  tannin  in  ether,  -         20  c.c. 

5  per  cent,  aqueous  solution  of  pure  iron-alum,  -  10     ,, 

Saturated  solution  of  fuchsin  in  90  per  cent,  alcohol,  -  1     „ 


152  FLAGELLUM   STAINING 

Pour  these  solutions  into  a  well  plugged  flask :  heat  the  mixture  to  100°  C.  in  a 
water  bath,  thoroughly  mixing  the  ingredients  by  shaking  and  continue  to  heat 
until  the  liquid  is  pale  in  colour.  The  solution  must  be  kept  in  an  hermetically 
sealed  bottle. 

1.  Mordanting. — Flood  the  cover-glass  with  a  few  drops  of  the  mordant  for  2  or 
3  minutes  at  25°  C.  or  10  minutes  at  15°  C.     Wash  in  water.     Dry. 

2.  Staining. — Stain   in  the  following  solution  for  a  few   seconds,   heating   the 
solution  until  steam  just  begins  to  rise : 

Fuchsin,  0'25  gram. 

Absolute  alcohol,     -  -         10  c.c. 

Carbolic  acid  crystals,      -  5  grams. 

Distilled  water,        -  -       100  c.c. 

3.  Mounting. — Wash  in  water.  Dry.     Mount  in  balsam. 

(vii)   Fitfield's  method.    Benignetti  and  Gino's  method. 

In  Pitfield's  method  the  mordant  and  the  stain  are  combined  in  one 
solution,  thus  : 

Saturated  aqueous  solution  of  alum,  -         10  c.c. 

10  per  cent,  solution  of  tannin  in  water,     -  -         10     „ 

Saturated  alcoholic  solution  of  gentian-violet,     -  2     „ 

Benignetti  and  Gino  obtain  very  satisfactory  results  with  the  following 
very  simple  method  which  is  a  modification  of  the  above. 

The  combined  mordanting  and  staining  solution  is  prepared  thus  : 


A.  Zinc  sulphate, 
Tannin, 
Distilled  water, 

B.  Solution  A,     - 

Saturated  aqueous  solution  of  alum, 
Saturated  alcoholic  solution  of  gentian-violet, 


1  gram. 
10  grams. 
100       „ 

5  c.c. 
5    „ 
3    , 


Technique. — Fix  the  film  with  heat,  and  when  cool  flood  it  with  a  large 
drop  of  solution  B,  and  heat  it  over  the  pjrot  light  of  a  Bunsen  until  steam 
just  begins  to  rise.  Wash  in  water.  Dry.  Mount  in  balsam. 

[R.  Muir's  modification  of  Pitfield's  method. 

[Prepare  : 

A.  Filtered  10  per  cent,  aqueous  solution  of  tannin,  -         10  c.c. 

Saturated  aqueous  solution  of  perchloride  of  mercury,  5     „ 

Saturated  aqueous  solution  of  alum,  5     „ 

Carbol-fuchsin,        -  5    „ 

[Mix  thoroughly.  Allow  the  precipitate  to  settle.  Decant  off  the  clear 
supernatant  fluid.  The  mordant  will  keep  for  about  a  fortnight. 

B.  Saturated  aqueous  solution  of  alum,  -         10  c.c. 

Saturated  alcoholic  solution  of  gentian- violet,     -  2     „ 

The  stain  does  not  keep. 

[1.  Prepare  and  fix  a  film  as  above  (p.  149). 

[2.  Flood  the  preparation  with  the  mordant  and  heat  until  steam  just 
begins  to  rise.  Let  the  solution  act  for  1  minute. 

[3.  Wash  thoroughly  in  running  water.     Blot  and  dry  over  the  flame. 

[4.  Flood  the  film  with  the  stain,  heat  as  before  for  1  minute. 

[5.  Wash  well  in  water. 

[6.  Blot.     Dry.     Mount  in  balsam.  ] 

(viii)   Bowhffl's  method. 

This  method  is  troublesome  and  offers  no  special  advantages. 

1.  Mordanting. — Place  the  film  for  10  minutes  at  a  temperature  of  40°-50°  C.  hi  a 


FLAGELLUM  STAINING  153 

bath  consisting  of  equal  parts  of  the  following  solutions  mixed  just  before  use  and 
filtered  : 

SOLUTION  A. 

Orcein,  -  1  gram. 

Absolute  alcohol,     -  -        50  c.c. 

Distilled  water,        -  -         40     „ 

SOLUTION  B. 

Tannin,  -  8  grams. 

Distilled  water,        -  -        40  c.c. 

Use  heat  to  dissolve  the  tannin. 

In  staining  the  flagella  of  the  Vibrio  cholerce  asiaticce  add  1  c.c.  of  a  saturated 
solution  of  alum  for  each  10  c.c.  of  mordant. 

2.  Washing. — Wash  in  water.     Dry. 

3.  Staining. — Flood  the  film  with  aniline-gentian-violet  and  heat   until  steam 
just  rises  from  the  film  for  15-30  seconds. 

4.  Mounting. — Wash.     Dry.     Mount  in  balsam. 

(ix)  Gemelli's  method. 

1.  Immerse  the  films  in  the  following  solution  for  10-20  minutes. 

Potassium  permanganate,         -         -  0'25  gram. 

Distilled  water,        -  -       100  c.c. 

2.  Wash  in  distilled  water. 

3.  Stain  for  15-30  minutes  in  the  following  mixture  : 

0'75  per  cent,  aqueous  solution  of  calcium  chloride,     -         -         20  c.c. 
1  per  cent,  aqueous  solution  of  neutral-red,         -         -  1     „ 

4.  Wash  in  water.     Dry  in  the  air.     Mount  in  balsam. 

(x)   Sclavo's  method. 

Sclavo's  method  fails  to  stain  the  flagella  of  some  micro-organisms  especi- 
ally the  flagella  of  the  cholera  vibrio.  The  author's  experience  has  been 
that  it  is  equally  unsuited  for  the  flagella  of  the  colon  bacillus. 

1.  Flood  the  film  with  a  large  drop  of  the  mordant,  viz. : 

50  per  cent,  alcohol,         .....  .       100  c.c. 

Tannin,  1  gram. 

Leave  for  1  minute  and  then  wash  in  water. 

2.  Treat  for  1  minute  with  the  following  solution  on  the  film  : 

Phospho-tungstic  acid,     -------          5  grams. 

Water,  -  -         -       100  c.c. 

3.  Wash  quickly  in  water. 

4.  Stain  for  3  or  5  minutes  with  a  drop  of  aniline-gentian-violet  heating  the 
stain  until  steam  rises  gently  from  the  film. 

5.  Wash  and  examine  in  water.     Dry.     Mount  hi  balsam. 

|  (xi)  Stephens'  method.] 
(Method  recommended.) 

[The  method  worked  out  by  J.  W.  W.  Stephens  is  a  modification  of  van 
Ermengem's  (p.  149)  and  depends  upon  the  use  of  very  strong  ammonia  as 
the  reducing  agent.  With  ordinary  care  a  satisfactory  result  can  be  absolutely 
relied  upon. 

[To  clean  the  slides. — Rub  the  slides  with  a  clean  cloth,  place  them  on  a  piece  of 
clean  wire  gauze  and  heat  with  a  smokeless  flame  for  some  minutes  (by  this  means 
grease  is  completely  removed).  Leave  the  slides  until  cool. 

[To  prepare  the  film. — Rub  a  little  of  the  culture  in  a  small  drop  of  tap-water  in 
a  watch-glass.  Transfer  a  drop  with  a  very  small  platinum  loop  to  a  minute  drop 
of  water  on  the  slide.  Mix.  Spread  with  the  loop  as  quickly  as  possible.  The 
film  should  dry  immediately  if  only  a  small  drop  of  water  has  been  used.  A 


154  EXPERIMENTS   ON   MOTILITY 

twenty-four  hour  growth  on  agar  does  quite  well  (a  younger  one  is  perhaps  better, 
but  flagella  can  be  shown  for  a  week  or  fortnight  or  more). 

[The  following  solutions  are  required  : 

(a)  The  mordant. 

2  per  cent,  aqueous  solution  of  osmic  acid,       ...  1  part. 

20  per  cent,  aqueous  solution  of  tannin,  3  or  4  parts. 

(b)  Silver  nitrate  solution. 

Crystals  of  silver  nitrate,         -         -  1  gram. 

Distilled  water, 100  c.c. 

(c)  Reducing  solution. 

2  per  cent,  aqueous  solution  of  gallic  acid,        -         -  1  part. 

Ammonia  fort.,1      --------  1     ., 

Mix  immediately  before  use. 

[1.  Place  the  mordant  on  the  film  for  one  or  two  minutes  or  less  (time 
unimportant). 

[2.  Wash  in  tap-water  thoroughly.     Shake  off  as  much  water  as  possible. 

[3.  Place  a  few  drops  of  the  silver  nitrate  solution  on  the  film  for  a  few 
seconds  or  longer. 

[4.  Shake  off  the  excess  of  silver  solution. 

[5.  Allow  one  drop  of  the  reducing  solution  to  fall  on  the  middle  of  the  film 
from  a  pipette.  A  wave  spreads  away  from  the  centre  to  each  end  of  the 
slide.  As  soon  as  the  film  is  seen  standing  out  clearly  and  black  (a  few 
seconds),  wash  off  in  tap- water. 

[6.  Pour  another  drop  or  two  of  the  silver  solution  on  to  the  film  and  leave 
for  half  a  minute  or  so. 

[7.  Wash  in  tap-water.  Blot.  Dry  over  a  flame.  The  preparations  fade 
rapidly  if  mounted  in  balsam  or  cedar- wood  oil.] 

SECTION  IV.— METHODS   OF  STUDYING  THE  MOTILITY   OF 
MICRO-ORGANISMS. 

Closely  connected  with  the  morphological  study  of  the  flagella  of  micro- 
organisms is  the  investigation  of  their  motility.2  Motile  organisms  can  make 
their  way  through  porous  substances  such  as  sand  or  a  filter  composed  of 
porcelain  or  siliceous  earth.  The  time  occupied  in  traversing  a  given  thick- 
ness of  sand,  etc.,  will  vary  according  as  to  whether  the  organism  is  actively 
or  feebly  motile. 

These  observations  are  utilized  for  determining  whether  or  no  an  organism 
is  motile,  for  separating  motile  from  non-motile  species,  for  determining  the 
relative  motility  of  different  strains  of  the  same  organism,  and  even  for 
creating,  by  a  process  of  selection,  races  which  are  endowed  with  exceptional 
powers  of  movement. 

A. — Cambier  has  drawn  attention  to  the  property  possessed  by  the  typhoid 
bacillus  of  traversing  the  walls  of  porous  structures,  and  has  suggested  that 
this  property  might  be  made  use  of  in  attempting  the  isolation  of  the  organism. 

A  porous  porcelain  bougie  is  placed  in  a  large  test-tube,  and  both  the 
bougie  and  the  test-tube  are  half-filled  with  broth ;  the  tube  is  plugged 
with  wool  and  the  whole  apparatus  autoclaved.  When  cool,  the  broth  in 
the  bougie  is  sown  with  a  culture  of  the  typhoid  bacillus.  After  incubating 
for  a  few  hours  at  37°  C.  the  broth  in  the  tube  surrounding  the  bougie  will 
be  distinctly  cloudy,  and  this  is  due  to  the  fact  that  the  typhoid  bacillus 

1  [It  is  essential  that  the  solution  of  ammonia  be  the  strongest  obtainable.] 
zVide  also  Chap.  VII.,  Dark-ground  illumination. 


EXPERIMENTS   ON  MOTILITY 


155 


has  passed  through  the  porous  walls  of  the  bougie.  Only  motile  organisms 
can  do  this,  and  of  these  the  typhoid  bacillus  is  one  of  the  first  to  pass  through. 
In  attempting  the  isolation  of  the  typhoid  bacillus  from  water,  the  broth  in 
the  bougie  would  be  sown  with  some  of  the  suspected  water,  and  when  the 
broth  surrounding  it  became  cloudy  a  small  quantity  would  be  removed  for 
the  purposes  of  further  investigation  by  the  usual  methods  (Chap.  XXIII.  ). 

B.  —  Carnot  and  Gamier  conceived  the  idea  of  making  motile  organisms 
pass  by  their  own  efforts  through  a  layer  of  sand  of  known  thick- 
ness, and  then  collecting  the  first  organisms  to  pass  through  ; 
they  were  thus  able  to  determine  exactly  the  time  required  by 
a  given  organism  to  make  its  way  through  a  given  thickness  of 
sand.  The  degree  of  motility  possessed  by  any  species  of  micro- 
organism can  by  these  means  be  exactly  measured. 

Technique.  —  1.  A  piece  of  glass  tubing,  7  mm.  calibre,  is 
drawn  out  in  the  flame  about  its  middle,  and  bent  into  an 
U  -shape  with  the  two  limbs  parallel  and  closely  applied  to  each 
other,  each  being  about  25  cm.  long  (fig.  128). 

2.  A  loosely-packed  plug  of  glass  wool  C  is  pushed  down  the 
limb  A  as  far  as  the  constriction  in  the  lower  part.     Broth  is 
then  poured  in  to  a  depth  of  about  10  cm.  in  each  tube.     Very 
fine  quartz  sand  (previously  washed  in  hydrochloric  acid  for 
48  hours,  and  then  in  water  for  several  days  and  afterwards 
calcined  in  the  hot  air  sterilizer)  is  slowly  dropped  down  the 
tube  A  until  it  forms  a  column  10-15  cm.  high.     A  and  B  are 
then  plugged  with  wool,  and  the  tube  autoclaved. 

3.  The  organism  whose  motility  is  to  be  investigated  is  then 
sown  in  the  broth  contained  in  the  limb  B  in  which  there  is 
no  sand,  and  the  tube  incubated  at  37°  C.     The  passage  of 
organisms  through  the  sand  is  made  manifest  by  a  cloudiness 

of  the  broth  in  A.  Only  motile  organisms  reach  the  broth  in  A,  and  the  time 
occupied  varies  with  different  species. 

Carnot  and  Garnier  give  the  following  times  for  the  most  motile  organisms  : 
Vibrio  cholerse  (Massaouah)  traverses  1  c.c.  of  sand  in  1  hr.  38  m. 
„  ,,     (Dantzig)  ,  2  hrs.  4  m. 


FIG.    128.— 


(Paris,  1884) 
Bacillus  psittacosis 
Bacillus  febris  entericae 
Bacillus  coli  communis 


4  hrs. 

2  hrs. 

3-6  hrs. 

(variable,    1    hr.   to 

several  days). 

Streptococci  with  feeble  undulatory  movements,  4  hrs.  50  m. 

Bacillus  anthracis,  Staphylococcus  pyogenes,  Pneumococcus,  etc.,  do  not  pass  through 
the  sand. 

This  method  like  that  of  Cambier  may  be  utilized  for  the  isolation  of 
motile  organisms ;  moreover  it  renders  possible,  by  successive  passages 
of  selected  organisms,  the  creation  of  strains  of  a  given  bacillus  possessed  of 
exceptional  motility.  In  this  way  Carnot  and  Garnier  were  able  after  five 
passages  to  isolate  from  a  culture  of  the  typhoid  bacillus,  which  originally 
took  6  hours  to  traverse  a  centimetre  of  sand,  a  strain  which  passed  through 
the  same  thickness  in  1  hour  and  4  minutes. 


CHAPTER   X. 
ANIMAL   INOCULATION. 

Section  I. — The  selection  of  animals  for  inoculation,  p.  156. 

Section  II. — The  keeping  of  animals,  p.  157. 

Section  III. — The  spontaneous  diseases  of  experimental  animals,  p.  159. 

Section  IV. — The  handling  of  experimental  animals,  p.  160. 

Section  V. — Experimental  inoculations,  p.  165. 

1.  Instruments,  p.  165.     2.  Preparation  of  the  material  for  inoculation,  p.  169. 
3.  Technique  of  inoculation,  p.  170. 
Section  VI. — Observations  to  be  made  on  inoculated  animals,  p.  182. 

SECTION  I.— THE  SELECTION  OF  ANIMALS  FOR  INOCULATION. 

FOR  purposes  of  experimental  inoculation,  animals  are  chosen  preferably 
from  among  the  mammalia,  less  frequently  from  among  the  other  vertebra ta. 
In  deciding  upon  what  species  of  animal  shall  be  used  for  a  given  experiment 
there  are  of  course  various  considerations  which  must  be  taken  into  account. 

1.  Susceptibility. — In  the  first  place  it  is  obviously  necessary  to  select  a 
species  of  animal  suitable  for  the  experiment  in  view.     To  produce  a  given 
disease  experimentally,  an  animal  susceptible  to  the  virus  should,  generally 
speaking,  be  chosen,  though  it  is  sometimes  desirable  to  use  an  animal  immune 
to  the  particular  disease  and  to  destroy  its  immunity  in  some  way  or  another. 
Some  knowledge,  therefore,  of  the  diseases  to  which  animals  available  for 
experimental  purposes  are  susceptible  is  more  or  less  indispensable.     In 
subsequent  chapters  those  animals  which  are  susceptible  to  the  action  of  the 
principal  micro-organisms  will   be   mentioned.     When   a   new  organism  is 
under  investigation  and  its  pathogenic  properties  have  to  be  determined  it  is 
desirable  to  inoculate  as  many  different  species  of  animals  as  possible. 

2.  Economic  considerations. — In  the  majority  of  cases  small  animals  are 
used  ;    they  are  cheap  to  buy,  and  can  be  kept  and  fed  at  small  expense, 
and,  if  need  be,  can  be  bred  in  the  laboratory. 

3.  General  considerations. — Whenever  possible  animals  of  quiet  habits  are 
chosen  because  they  are  easy  to  handle,  and  do  not  require  elaborate  cages. 

Small  rodents,  such  as  rabbits,  guinea-pigs,  white  mice,  white  rats,  common 
brown  mice  [Mus  musculus],  and  house  rats  [Mus  decumanus]  are,  on  the 
whole,  more  often  used  than  any  other  animals  for  experimental  inoculation ; 
they  are  easily  obtained,  and  the  first  four — to  which  the  term  "  laboratory 
animals  "  generally  refers — are  susceptible  to  most  of  the  organisms  patho- 
genic to  man. 

Cattle,  goats,  pigs,  horses,  sheep,  asses  and  birds  (fowls  and  pigeons)  are 
also  used  for  experiment  in  special  cases. 


THE   SELECTION   OF   ANIMALS 


157 


Cats  are  difficult  to  handle,  and  dogs  are  only  slightly  susceptible  to  most 
of  the  organisms  pathogenic  to  man. 

Frogs  are  occasionally  used,  but  they  are  not  very  susceptible. 

Ground  squirrels  [Mus  citullus  ]  are  not  only  difficult  to  get  in  this  country, 
but  they  do  not  breed  in  captivity. 

Monkeys,  and  especially  the  anthropoid  apes,  have  for  some  time  been 
little  used  for  experimental  purposes  on  account  of  the  difficulty  of  obtain- 
ing them,  their  initial  cost,  and  the  great  care  with  which  they  have  to  be 
tended  in  captivity.  Nevertheless,  the  work  of  MetchnikofE  and  his  pupils 
on  syphilis,  [of  the  English  Commission  on  tuberculosis,  of  Levaditi  and 
Landsteiner  on  acute  polio-myelitis,  etc.  ]  has  shown  the  value  of  experiments 
upon  these  animals  in  the  investigation  of  human  disease.  [Macacus  rhesus 
is  the  most  suitable — for  most  purposes — and  at  the  same  time  the  cheapest 
monkey.  ] 


SECTION  II.— THE  KEEPING  OF  ANIMALS. 
A.  Small  animals. 

Accommodation. — The  "  small  animal "  house  ought  to  be  spacious,  well 
ventilated,  floored  with  concrete  or  some  similar  impervious  material  and 
have  water  laid  on,  so  that  it  can  be  frequently  washed  down. 

Animals  generally,  and  especially  monkeys,  rabbits,  mice  and  rats,  are 
very  susceptible  to  cold  and  damp  ;  the  animal  house  must,  therefore,  be 
kept  dry,  and  facilities  for  warming  it  in  winter  should  be  provided. 

Cages,  feeding,  etc. — The  cages  should,  as  far  as  possible,  be  made  of  metal. 
It  is  a  bad  practice  to  place  one  cage  on  top  of  another,  since  fluids  from  the 
upper  cage  may  soil  [and  infect]  the  one  beneath.  If,  from  want  of  space, 
it  becomes  absolutely  necessary  to  place  one  cage  on  top  of  another  there 
must  be  a  sheet  of  metal  between  them  tilted  and  guttered,  so  that  the 
urines  run  off.  The  bottom  of  the  cages  should  always  be  perforated. 

[A  rectangular  cage  made  entirely  of  fairly  stout  galvanized  iron  wire 
and  resting  on  a  metal  tray  is  both  efficient  and  cheap  (fig.  129). 


FIG.  129.— Animal  cage.     The  cage  is  raised  on  blocks  from  the  tray. 

[The  cage  itself  consists  merely  of  a  rectangular  wire  box  of  which  one  of 
the  two  largest  sides  is  hinged  to  form  a  lid  allowing  access  to  the  interior. 
A  clasp  must  also  be  provided  so  that  the  top  can  be  securely  fastened  down. 
The  cage  is  placed  on  a  sheet  of  galvanized  iron  turned  up  to  the  extent  of 
1  or  1J  in.  all  round  to  make  it  water-tight  and  measuring  1  inch  larger  in 


158  THE   KEEPING   OF   ANIMALS 

each  direction  than  the  cage.  To  clean,  it  is  only  necessary  to  lift  the  cage 
off  the  tray.  It  is  an  advantage  to  have  two  trays  for  each  cage  so  that  one 
may  be  disinfected  and  dried  while  the  other  is  in  use.] 

The  cages  must  be  cleaned  daily,  and  should  an  animal  die  or  be  removed 
to  make  room  for  a  new  occupant  the  cage  in  which  it  lived  must  be  dipped 
in  some  strong  antiseptic  solution  (phenol,  lysol)  or  be  sterilized 
by  flaming  it  with  spirit  or  with  a  large  specially  constructed  gas 
burner  (fig.  130). 

Each  cage  should  carry  a  label  indicating  the   nature   of  the 
experiment,  and  the  day  upon  which  the  animal  was  inoculated. 
(i)  Rabbits,  guinea-pigs,  rats  and  mice. — Rabbits  and  guinea-pigs 
can  be  conveniently  kept  in  the  cages  described  above. 

Grey  rats  [Mus  decumanus],  as  well  as  house  [Mus  musculus] 
and  field  [Mus  silvaticus]  mice  should,  as  a  rule,  be  caged  singly. 
When  several  of  these  rodents  are  put  in  one  cage  they  fight,  and 
frequently  kill  each  other.  They  are  best  kept  in  large  wide- 
mouthed  jars,  the  mouth  being  covered  with  metal  gauze  fastened 
down  round  the  neck  with  iron  wire. 

White  rats  are  frequently  very  tame,  and  can  be  kept  in  small- 
FIG  130      mesn  wire-netting  cages  or  bird  cages,  or  even  in  wooden  boxes  fitted 
GasIGburiier  with  a  wire-netting  door. 

ca,r  e?aming  White  mice  should  be  kept  in  glass  jars  or  metal  boxes  such  as 
Palmer's  biscuit  boxes,  the  lid  of  which  must  be  pierced  with  a 
number  of  holes.  The  floors  of  the  boxes  or  jars,  whichever  be  used,  should 
be  covered  with  a  layer  of  sawdust  several  centimetres  deep,  and  a  little  wool 
should  also  be  put  in  the  cage  as  mice  do  not  like  the  cold.. 

There  is  no  need  here  to  discuss  the  proper  feeding  of  rabbits,  guinea- 
pigs,  etc. 

Mice  and  rats  should  be  fed  on  corn  and  moistened  bread.  White  rats 
are  very  fond  of  water,  and  a  small  dish  containing  it  should,  therefore,  be 
put  in  their  cages. 

(ii)  Monkeys. — Monkeys  require  a  great  deal  of  attention.  Their  cages  must 
be  large  and  be  kept  scrupulously  clean.  They  are  very  susceptible  to  the 
cold,  and  the  house  in  which  they  are  kept  ought,  in  these  climates,  to  be  artifi- 
cially heated  most  of  the  year.  The  nature  of  the  food  required  varies  with 
the  different  species,  but,  generally  speaking,  milk,1  dried  fruits,  [cooked  rice], 
bananas  and  bread  constitute  the  staple  articles  of  diet  of  monkeys  in  cap- 
tivity. They  should  be  given  something  to  drink  twice  a  day,  but  it  is 
advisable  not  to  leave  water  [or  milk]  in  their  cages. 

(iii)  Frogs. — Frogs  can  be  easily  kept  at  ordinary  room  temperatures,  but 
at  temperatures  approximating  to  that  of  the  human  body,  such  as  are 
sometimes  necessary  under  experimental  conditions,  they  often  die  in  a  day 
or  two  without  any  apparent  reason. 

Ledoux-Lebard  suggests  the  following  as  a  useful  method  for  keeping  frogs  (Rana 
esculenta  is  better  than  Rana  temporaria)  at  a  temperature  of  35°  or  37°  C.  for  a 
month  or  more.  Keep  each  frog  in  a  bottle  containing  a  few  centimetres  of  water 
and  covered  with  a  piece  of  stout  muslin  tied  on  with  string,  renew  the  water  daily 
with  a  fresh  supply  at  the  same  temperature,  and  cram  the  frog  once  a  week  with 
beef,  veal  or  mud  worms. 

Isolation  of  inoculated  animals. — Inoculated  animals  must,  of  course,  be 
rigidly  kept  away  from  the  neighbourhood  of  normal  animals. 

[x  It  must  not  be  forgotten  that  monkeys  and  apes  are  highly  susceptible  to  tuberculosis, 
so  that  the  milk  must  either  come  from  an  unimpeachable  source  or  be  sterilized  before 
being  fed  to  them.] 


THE   SPONTANEOUS   DISEASES   OF  ANIMALS  159 

Breeding  of  small  animals. — Rabbits,  guinea-pigs,  white  rats,  and  white 
mice  can  easily  be  bred  in  the  laboratory,  and  because  of  their  inclination  to 
kill  the  newly-born  animals,  it  is  as  well  to  separate  the  males  from  pregnant 
females.  This  precaution  is  absolutely  necessary  in  the  case  of  rabbits  and 
mice,  but  is  less  important  with  white  rats  and  perhaps  unnecessary  for 
guinea-pigs. 

B.  Large  animals. 

[With  regard  to  the  housing  of  large  animals  such  as  cattle,  sheep,  pigs, 
etc.,  it  is  unnecessary  to  say  more  than  that  the  houses  or  pens  should  be 
designed  on  lines  similar  to  those  in  which  they  are  ordinarily  stabled.  The 
structures  should  be  light  and  well  ventilated  the  floors  concreted  or  cemented 
and  well  drained.  The  walls  should  be  constructed  of  material  which  readily 
lends  itself  to  efficient  cleansing  with  antiseptics.  If  the  stalls  be  of  wood 
they  should  be  limewashed  out  with  lime  mixed  with  2  per  cent,  lysol  before 
a  new  animal  is  introduced.] 

SECTION  III.— THE  SPONTANEOUS  DISEASES   OF  EXPERIMENTAL 

ANIMALS. 

Laboratory  animals  are  liable  to  certain  infectious  diseases  with  the  more 
common  of  which  it  is  important  to  be  familiar  because  they  are  sometimes 
responsible  for  a  heavy  mortality  among  experimental  animals. 

Abscess. — Large  abscesses  containing  thick,  fetid  pus  not  infrequently 
occur  in  rabbits  in  various  parts  of  the  body.  They  lead  to  a  cachectic 
condition,  and  ultimately  end  in  death.  The  disease  is  contagious. 

The  infected  animal  must  be  isolated  and  the  cage  carefully  disinfected. 
Treatment  consists  in  opening  the  abscess,  evacuating  the  pus  and  gently 
curetting  the  wall,  subsequently  washing  it  out  at  frequent  intervals,  and 
dressing  it  writh  antiseptic  dressings. 

Acari. — An  acarus  sometimes  develops  in  the  external  auditory  meatus 
of  the  rabbit ;  it  soon  invades  the  middle  ear,  and  causes  serious  nerve  troubles, 
such  as  gyratory  movements,  convulsions  and  epileptiform  seizures  which 
lead  to  the  death  of  the  animal.  The  disease  may  be  recognized  by  the 
yellow  crusts  which  are  seen  in  the  rabbit's  ear  and  which,  if  examined 
microscopically  (Oc.  2,  obj.  A.  Zeiss),  are  found  to  be  composed  of  amor- 
phous debris  and  numerous  acari.  The  disease  is  highly  contagious  but 
yields  to  treatment  if  taken  in  hand  in  the  early  stages. 

Immediately  a  case  is  found  in  the  animal  house,  the  infected  individual 
should  be  killed,  unless  the  experiment  be  of  special  interest,  in  which  event 
it  must  be  isolated  and  treated.  Treatment  consists  in  washing  off  the 
crusts  formed  on  the  auditory  meatus  daily  with  a  sponge  made  by  twisting 
a  little  piece  of  wool  round  a  small  rod,  and  dropping  a  few  drops  of  a  O5  per 
cent,  solution  of  polysulphide  of  potassium  (liver  of  sulphur)  into  the  ear. 
The  infected  animal's  cage,  and  those  near  it,  should  be  disinfected  and  the 
ears  of  all  the  other  rabbits  in  the  house  frequently  examined. 

Septicaemias. — Rabbits  and  guinea-pigs  are  subject  to  epizootic  diseases 
which,  only  too  often,  decimate  the  population  of  the  animal  house  in  a 
few  days. 

As  a  rule,  rabbits  and  guinea-pigs  are  affected  at  the  same  time.  The 
animals  curl  themselves  up,  their  coats  are  rough,  and  they  suffer  from  a 
running  from  the  nose  and  diarrhoea.  Death  soon  follows  these  symptoms. 
Post  mortem,  lesions  of  broncho-pneumonia  are  seen.  The  disease  appears 
to  be  due  to  a  small  bacillus  morphologically  similar  to  Pfeiffer's  [influenza] 
bacillus. 


160  THE   HANDLING   OF  ANIMALS 

Pasteurellosis. — Another  disease,  caused  by  an  organism  of  the  Pasteurella 
group  (Chap.  XXVIII.),  is  sometimes  seen  among  rabbits.  Infection  is  due  to 
contamination  of  the  food  and  floors  of  the  cages  with  infected  excreta.  The 
animals  are  listless,  suffer  from  diarrhoea  and  succumb  rapidly.  Post  mortem  : 
excess  of  fluid  in  the  pleurae,  pericardium  and  peritoneum  ;  congestion  of 
the  lungs,  intestines,  etc. 

HfPhisalix  has  described  a  disease  caused  by  a  Pasteurella  similar  to  the 
canine  pasteurella  (Chap.  XXVIII.)  which  may  sometimes  produce  an 
epizootic  among  guinea-pigs. 

Certain  contagious  pneumonias  (Weber,  Tartakowsky  and  others)  may 
also  attack  laboratory  animals. 

When  a  septicsemic  infection  makes  its  appearance  in  the  animal  house, 
isolate  the  animals  which  are  obviously  or  suspected  to  be  infected  and 
disinfect  the  house.  It  is  even  better,  especially  if  there  be  any  which  it  is 
particularly  important  not  to  lose  (animals  undergoing  immunization,  etc.), 
to  remove  the  animals  which  are  healthy  to  some  other  place  altogether,  and 
to  transfer  them  to  disinfected  cages.  Still  it  will  often  be  difficult,  what- 
ever be  done,  to  prevent  the  spread  of  the  infection. 

[Several  epizootics  resulting  in  a  heavy  mortality  among  the  guinea-pigs 
in  the  small  animal  house  and  due  to  organisms  other  than  those  mentioned 
have  now  been  recorded.  From  the  internal  organs  bacilli  of  the  paratyphoid 
group  have  been  isolated  (B.  Gaertner,  M'Conkey,  Petrie,  Bainbridge  and 
O'Brien,  B.  aertrycke,  O'Brien,  Petrie  and  O'Brien).  There  is  evidence, 
however,  that  these  organisms  were  not,  at  least  in  some  of  the  epizootics, 
the  real  cause  of  the  disease,  which  appeared  to  be  a  filter-passing  organism 
(Petrie  and  O'Brien),  the  paratyphoid  bacilli  being  "  secondary  "  infections.] 

Goccidiosis. — Rabbits  are  frequently  infected  with  Coccidium  oviforme 
(vide  Sporozoa).  It  is  important  that  this  fact  be  kept  in  mind  and  it  should 
be  noted  that  the  disease  is  particularly  troublesome  in  young  animals. 

Numerous  other  parasites  may  be  found  in  experimental  animals,  and 
reference  will  be  made  to  these  in  due  course,  more  particularly  when  dealing 
with  Tuberculosis,  Glanders,  Pleuro-pneumonia,  the  Pasteurelloses,  the  Hsema- 
tozoa,  the  Trypanosomata,  etc. 

[Pseudo-tuberculosis. — Pseudo -tuberculosis  is  a  most  troublesome  disease 
among  guinea-pigs  and  rabbits  not  only  because  the  naked-eye  lesions  so 
closely  resemble  the  lesions  produced  by  the  tubercle  bacillus  but  because 
so  many  animals  become  infected  and  die  once  the  disease  makes  its  appear- 
ance in  the  animal  house.  Pseudo-tuberculosis  is  the  result  of  infection  with 
a  short  stout  bacillus  with  rounded  ends  which  grows  readily  on  agar  at  the 
temperature  of  the  body.  A  feature  which  may  arouse  suspicion  is  the  fact 
that  the  mesenteric  glands  are  markedly  affected  which  is,  of  course,  not  the 
case  when  an  animal  has  been  inoculated  sub-cutaneously  or  intra-peritoneally 
with  the  tubercle  bacillus.  Infection  apparently  takes  place  through  the 
alimentary  canal.  When  the  disease  appears  all  the  animals  in  the  animal 
house  must  be  isolated  and  as  many  as  possible  killed.  It  is  a  wise  thing  to 
destroy  any  food  or  bedding  in  stock  and  order  a  fresh  supply.  ] 

SECTION  IV.— THE  HANDLING   OF   EXPERIMENTAL  ANIMALS. 

Most  animals  struggle  when  they  are  caught  and  try  to  bite  or  scratch  the 
person  holding  them.  It  is  important  to  avoid  these  wounds,  which  may  be 
dangerous,  especially  when  the  animal  is  infected  with  a  disease  transmissible 
to  man,  e.g.  hydrophobia.  A  skilled  worker  should  never  be  damaged  by  the 
animals  he  handles. 


RABBITS  161 

During  an  experiment  the  animal  may  be  held  either  by  the  person  operating 
•or  by  an  assistant.  This  is  quite  satisfactory  in  the  case  of  most  animals  when 
a,  simple  sub-cutaneous  inoculation  has  to  be  done,  but  for  the  more  difficult 
operations,  such  as  inoculation  into  the  peritoneum,  meninges  or  veins,  or 
when  dangerous  viruses,  such  as  those  of  glanders,  hydrophobia,  etc.,  have 
to  be  inoculated,  it  may  be  better  to  hold  the  animal  in  some  suitable  form  of 
apparatus  designed  for  the  purpose.  [But  as  a  matter  of  fact  occasion 
for  the  use  of  such  apparatus  very,  very  seldom  arises.  If  there  is  likely  to 
be  any  difficulty  this  may  be  overcome  by  administering  an  anaesthetic. 
But  for  sub-cutaneous,  intra-peritoneal  or  intra-venous  inoculation  not  even 
an  anaesthetic  is  necessary.  ]  In  the  handling  of  small  animals  an  assistant 
should  be  dispensed  with  as  far  as  possible. 

1.  Rabbits. 

To  catch  a  rabbit  grasp  it  by  the  skin  of  the  back,  or  by  one  of  its  ears. 
These  are  the  only  ways  to  secure  the  animal  without  being  scratched  in  the 
attempt.  To  hold  a  rabbit,  place  the  animal  on  the  knees,  and  hold  it  there 
with  the  left  hand,  using  the  right  hand  for  the  inoculation.  If  the  animal 
be  troublesome,  take  hold  of  the  skin  of  the  back  with  the  right  hand,  and 
put  the  rabbit  under  the  left  arm,  so  that  the  head  and  fore  limbs  are  fixed 
between  the  arm  and  the  chest  wall,  support  the  trunk  on  the  forearm,  and 
hold  the  hind  limbs  with  the  left  hand.  The  right  hand  is  then  free  to  make 
the  inoculation. 

When  an  assistant  is  available  he  turns  the  rabbit  on  its  back  and  takes 
hold  of  the  four  limbs  in  his  left  hand,  holding  the  head  in  his  right  in  such  a 
way  that  the  top  of  the  animal's  head  rests  in  the  palm  and  his  thumb  passes 
under  the  lower  jaw. 

Apparatus  for  holding  rabbits. — A  rabbit  can  be  very  simply  held  by 
wrapping  it  up  to  its  neck  in  a  duster,  or  a  large  strip  of  cloth,  and  fastening 
the  limbs  beneath  the  body.  Operations  on  the  head  and  ears  can  then  be 
performed.  To  inoculate  one  of  the  limbs,  take  it  out  of  the  duster,  and 
hold  it  extended  in  the  left  hand. 

To  prevent  the  animal  moving  at  all,  several  pieces  of  apparatus  are  avail- 
able, for  example,  Malassez's,  Czermak's,  Piorkowski's,  Latapie's,  and 
Debrand's.1  The  two  latter,  which  may  be  used  for  all  the  smaller  animals, 
are  very  ingenious,  but  complicated  and  expensive.  Latapie's  apparatus 
is,  moreover,  difficult  to  disinfect. 

The  simplest  and  at  the  same  time  the  best  piece  of  apparatus  consists 
of  a  rectangular  sheet  of  zinc  or  copper,  the  edges  of  which  are  turned  up 
and  pierced  with  holes  2  or  3  cm.  apart.  Place  the  animal  on  the  metal  tray, 
put  a  noose  (of  string,  or,  better,  leather)  round  one  of  the  hind  limbs  and 
fasten  it  above  the  wrist,  pass  one  of  the  ends  through  a  hole  near  the  end  of 
the  tray  and  tie  it  to  the  other  end.  In  the  same  way  fasten  the  fore  limb 
of  the  opposite  side  to  a  hole  at  the  other  end  of  the  tray.  Then  make  the 
other  two  limbs  fast.  The  animal  is  now  absolutely  unable  to  move.  The 
head  can  be  held  by  an  assistant,  or  can  be  fixed  with  a  string  passing  from  a 
bar  introduced  behind  the  incisor  teeth,  and  fastened  as  before  to  two  holes 
of  the  tray. 

Or  the  head  can  be  held  with  a  Ranvier's  ring.  This  device  consists  of  an  horizontal 
iron  bar  moving  on  a  vertical  bar  by  means  of  a  double  joint,  which  allows  it  to  be 
fixed  in  any  position.  The  horizontal  bar  ends  in  a  ring  perpendicular  to  its  axis, 
and  on  to  the  ring  two  small  hooks  are  adjusted,  to  which  a  piece  of  elastic  can 
be  attached.  Fit  the  ring  on  the  animal's  nose  and  attach  the  elastic  to  one  of  the 

1Annales  de  VInstitut  Pasteur,  1894  and  1900. 


162  THE   HANDLING  OF  ANIMALS 

hooks,  then  pass  it  between  the  ears,  and  fasten  the  end  to  the  other  hook.  The 
apparatus  is  fixed  on  to  the  tray  by  means  of  a  screw  clamp.  The  animal  can  be 
secured  to  the  tray  and  its  head  held  by  the  ring,  either  on  its  back  or  belly  at  wilL 

Anaesthetics. — Rabbits  are  very  sensitive  to  anaesthetics.  Chloroform 
should  not  be  given  either  slowly  or  in  small  and  repeated  doses,  because  it 
will  thus  almost  certainly  kill  the  animal ;  but  by  giving  a  large  dose  to 
begin  with  and  then  stopping  the  administration  after  a  few  moments 
accidents  can  almost  always  be  avoided. 

Twist  two  or  three  thicknesses  of  filter  paper  into  a  cone,  pour  a  teaspoonful 
of  chloroform  on  to  it  and  hold  it  over  the  animal's  mouth.  Respiration 
stops  after  a  few  seconds  but  soon  begins  again  ;  at  this  moment  anaesthesia 
is  complete ;  the  administration  of  chloroform  should  now  be  stopped,  and 
the  Operation  quickly  performed. 

2.  Guinea-pigs. 

It  is  best  to  catch  guinea-pigs  by  the  skin  of  the  back  ;  they  are  easier  to 
handle  than  rabbits,  and  can  generally  be  held  in  the  left  hand,  leaving  the 
right  hand  free  for  inoculation. 

If  it  be  desired  to  hold  a  guinea-pig  with  instruments  the  simplest  way  is 
to  catch  hold  of  the  animal  by  the  skin  of  its  back  with  a  large  pair  of 
pressure  forceps,  the  grasping  ends  of  which  are  ring-shaped  (fig.  131).  The 


FIG.  131. — Forceps  for  grasping  small  animals. 

forceps  being  clipped  together  are  hung  by  one  of  the  finger  holes  on  a  nail  in 
the  wall,  and  the  animal  being  thus  suspended  is  rendered  quite  motionless. 

For  holding  guinea-pigs  still  while  taking  temperatures,  making  inocula- 
tions into  the  hind  limbs,  etc.,  it  will  be  found  convenient  to  secure  the 
anterior  part  of  the  animal  in  a  metal  cylinder  with  slits  along  the  sides. 

For  carrying  out  delicate  operations,  it  is  preferable  to  fix  the  animal  on 
the  tray  described  above.  Such  trays  should  be  kept  in  two  sizes,  the  larger 
ones  for  rabbits  and  the  smaller  for  guinea-pigs. 

Anaesthetics. — Guinea-pigs  are  less  susceptible  to  chloroform  than  rabbits. 
It  is  very  seldom  that  they  have  to  be  anaesthetized,  but  should  it  be  necessary 
to  give  a  guinea-pig  an  anaesthetic,  the  method  of  administration  is  the  same 
as  for  rabbits. 

3.  White  mice  and  white  rats. 

To  catch  the  animals. — These  species  are  perhaps  in  a  general  way  more 
used  for  experimental  inoculation  than  any  other  "  laboratory  animal." 
They  can  be  caught  by  the  tail  with  the  fingers.  Sometimes  they  struggle 
and  may  inflict  a  painful  bite  ;  this  can  be  avoided  by  grasping  them  by  the 
tail  or  skin  of  the  neck  with  a  pair  of  forceps. 

To  hold  them  for  inoculation,  the  only  method  that  can  be  recommended 
is  to  catch  hold  of  the  tail  with  the  fingers  or  a  pair  of  forceps  and  draw  it 
out  of  the  pot,  the  animal  being  thus  suspended  head  downwards  inside  ; 
then,  as  a  precaution  against  being  bitten  put  a  small  piece  of  board  over  the 


RATS— DOGS  163 

mouth  of  the  jar  so  that  only  the  tail  projects.  Inoculation  can  now  be 
made  at  the  root  of  the  tail.  If  it  is  necessary  to  inoculate  into  one  of  the 
limbs,  pull  the  limb  out  of  the  jar  with  a  pair  of  forceps.  [The  inoculation 
can  however  be  performed  in  the  following  manner  which  is  we  think  simpler 
than  that  just  described.  Let  an  assistant  catch  the  rat  by  the  tail  and  hold 
the  hind  limbs  with  one  hand  and  the  fore  limbs  with  the  other  at  the  same 
time  wrapping  a  small  cloth  loosely  round  the  animal's  head.  The  inoculation 
can  now  be  made  by  the  operator  into  any  part  of  the  animal's  body.  If  the, 
rat  bites  it  does  not  damage  the  cloth.] 

Anaesthesia. — For  all  delicate  operations  it  is  better  to  anaesthetize  the 
animal.  Rats  and  mice  are  easily  killed  by  chloroform,  but  take  ether 
well. 

Put  the  animal  under  a  bell  jar  with  a  small  piece  of  absorbent  wool  soaked 
in  ether  ;  or  the  wool  can  be  put  straight  into  the  jar  in  which  the  animal 
is  living.  When  it  falls  over  motionless  take  it  out  of  the  bottle  and  fix 
it  on  a  small  tray  ;  in  the  case  of  rats,  if  necessary,  put  on  a  Ranvier's  rat- 
bit  as  well,  or  use  a  Debrand's  apparatus.  Anaesthesia  can  be  prolonged  by 
giving  a  little  ether  from  time  to  time. 

4.  Grey  rats. 

Gre"y  rats  [Mus  decumanus]  struggle  vigorously  and  may  give  very  nasty 
bites.  They  can  only  be  caught  with  a  pair  of  large  strong  forceps,  such  as 
those  described  above  (fig.  131). 

Pass  the  forceps  into  the  bottle  containing  the  rat  and  catch  hold  of  the 
animal  quickly  wherever  it  is  possible  to  do  so.  The  rat  at  once  attacks  the 
forceps  and  bites  them  ;  while  the  animal  is  thus  engaged,  fix  a  second  pair 
of  forceps  on  to  the  skin  of  the  neck,  clamp  the  two  pairs  of  forceps  firmly 
and  lift  the  animal  out  of  the  bottle. 

During  the  inoculation,  an  assistant  holds  the  rat  securely  with  the  two 
pairs  of  forceps,  inclining  the  forceps  fastened  to  the  neck  towards  the  ver- 
tebral column  in  order  to  pick  up  the  tail  with  the  same  hand.  With  the 
other  hand  he  takes  the  other  pair  of  forceps  and  pulls  on  them  gently  so  as 
to  make  it  impossible  for  the  animal  to  use  his  teeth.  If  this  second  pair 
of  forceps  was  badly  fixed,  the  skin  over  the  lower  jaw  should  be  held  with 
another  pair.  When  the  operation  is  done  place  the  rat  safely  inside  the 
bottle  again  before  releasing  the  forceps. 

Anaesthesia. — Grey  rats  should  always  be  anaesthetized  before  beginning 
a  difficult  or  dangerous  inoculation.  Put  a  pledget  of  wool  soaked  in  ether 
in  the  bottle,  and  proceed  as  already  described  in  the  case  of  white  rats. 

5.  Dogs. 

If  the  dog  be  a  quiet  animal  catch  hold  of  him  firmly  by  the  skin  of  the 
neck.  When  dealing  with  a  surly  or  snappy  dog  use  a  special  pair  of  long 
iron  forceps  (pince  a  collier),  which,  when  closed,  form  a  collar  round  the 
animal's  neck.  Alternatively,  throw  a  noose  round  the  animal's  neck  and 
fasten  the  loose  end  to  a  bar  of  the  cage  or  a  post ;  as  the  dog  pulls  the  noose 
tightens,  the  animal  falls  over  half  suffocated,  and  the  opportunity  is  taken 
to  slip  on  a  muzzle  and  tie  its  feet. 

Muzzling. — No  operation  should  be  performed  on  a  dog  without  muzzling 
it  beforehand.  The  simplest  method  is  to  pass  a  piece  of  stout  string  into 
the  animal's  mouth  behind  the  canine  teeth,  make  a  simple  knot  below  the 
jaw,  bring  the  two  ends  up  and  tie  them  in  a  double  knot  on  the  nose.  Or, 
after  passing  a  stout  round  iron  wire  behind  the  canine  teeth,  take  two  turns 


164  THE   HANDLING   OF   ANIMALS 

with  a  piece  of  string  round  the  muzzle  behind  the  bit,  tighten  the  ligature 
and  fasten  the  ends  securely. 

If  it  be  necessary  to  gag  the  animal  with  the  mouth  open  for  the  purpose 
of  passing  a  catheter  into  the  stomach,  use  a  Claude  Bernard's  bit  with  a 
double  transverse  branch. 

Instead  of  the  bit  a  rectangular  wooden  gag  of  a  size  suitable  to  the  animal, 
and  pierced  with  a  hole  in  the  centre  may  be  used.  After  placing  this  gag  in 
the  mouth  behind  the  canine  teeth  fix  the  jaws  with  string. 

Method  of  holding  dogs. — After  muzzling,  the  dog  can  generally  be  held 
with  the  hands.  For  long  operations  the  animal  should  be  held  with  Claude 
Bernard's,  Malassez's,  or  Debrand's  apparatus,  or  more  simply  by  fixing  it 
by  the  feet,  as  has  been  described  in  the  case  of  the  rabbit,  on  to  a  heavy 
wooden  table  perforated  with  holes  or  fitted  with  hooks  through  which 
strings  can  be  passed. 

Anaesthesia. — It  is  rarely  necessary  to  anaesthetize  dogs  in  bacteriological 
work.  The  animals  take  chloroform  well,  provided  that  large  doses  are 
not  given,  and  the  liquid  does  not  come  in  contact  with  the  nasal  mucous 
membrane. 

In  giving  an  anaesthetic  a  long  muzzle  is  generally  used,  ending  in  a  small 
perforated  box  in  which  a  sponge  soaked  in  chloroform  is  placed.  The 
administration  can  be  suspended  or  continued  at  will  by  taking  off  or  replacing 
the  box  on  the  muzzle.  Small  doses  should  be  used  to  begin  with.  Anaes- 
thesia is  complete  after  8-15  minutes. 

6.  Cats. 

Cats  are  very  difficult  to  manage,  and  are  rarely  used  for  bacteriological 
experiments.  It  is  best  to  take  hold  of  them  firmly  by  the  skin  of  the  back  ; 
or,  if  the  cat  be  wild,  adopt  the  noose  method  described  above  for  dogs. 

In  operating  on  a  cat  it  is  well  to  anaesthetize  it.  As  soon  as  it  has  been 
caught,  put  it  into  a  large  wide-mouthed  jar  in  which  there  is  a  sponge 
soaked  in  chloroform,  and  cover  the  jar  at  once.  Cats  are  very  sensitive  to 
chloroform,  and  the  animal  must  be  taken  out  of  the  jar  as  soon  as  it  falls  : 
anaesthesia  will  continue  for  several  minutes  without  any  further  administra- 
tion. The  animal  may  either  be  fixed  on  to  the  table  already  described,  or 
may  be  wrapped  up  in  a  large  duster  with  the  feet  under  the  belly,  the 
head  and  anterior  part  of  the  body  being  pushed  into  a  sack.  This  is  an 
excellent  method  for  inoculations  into  the  posterior  part  of  the  body,  rectal 
injections,  etc. 

7.  Monkeys. 

Monkeys  especially  Macacus  rhesus  are  also  difficult  to  manage  [and  are 
so  active  that  it  is  hard  to  catch  them,  if  their  cage  be  at  all  large.  Wear 
rough  leather  gloves  and  grasp  the  animal  by  the  body  or  limbs,  then  take  a 
fresh  grip,  of]  the  skin  at  the  back  of  the  neck,  and  treat  them  in  the  same 
way  as  dogs.  It  is  necessary  to  chloroform  them  if  the  operation  is  likely 
to  last  any  length  of  time. 

8.  Horses  and  Asses. 

Horses  can  nearly  always  be  inoculated  without  adopting  any  special 
method  for  holding  them.  It  is  enough  for  an  assistant  to  hold  them  with  a 
bridle  or  halter.  If  a  horse  be  nervous  its  eyes  may  be  covered,  and  should 
it  struggle,  a  twitch  may  be  used  or  one  of  its  fore  legs  flexed  and  fastened. 
For  longer  operations  the  horse  may  be  shackled  and  thrown  by  methods 


CATTLE 


165 


well  known  to  people  who  have  to  do  with  horses.     Vinsol's  apparatus  is  to 
be  strongly  recommended,  but  unfortunately  it  is  very  expensive. 

9.  Cattle. 

Bovine  animals  are,  as  a  rule,  easily  managed.  For  long  operations  the 
animal  is  thrown  on  a  vaccination  inoculation  table,  or  placed  in  a  Vinsol's 
apparatus. 

10.  Birds. 

Fowls  and  other  birds  ordinarily  used  for  inoculation  are  easily  held  with 
the  hand.  They  may  be  secured  by  their  feet  and  wings  on  the  metal  tray 
described  on  p.  161,  or  on  a  Debrand's  apparatus. 

Note. — After  every  operation,  all  bits,  gags,  dishes,  etc.,  which  have  been 
used  must  be  carefully  cleaned  and  washed  with  a  solution  of  carbolic  acid, 
[lysol  or]  formalin,  and  the  antiseptic  washed  off,  of  course,  with  water  before 
the  instrument  is  used  again. 


SECTION  V.— EXPERIMENTAL   INOCULATIONS. 
1.   Instruments. 

There  is  no  need  to  go  into  details  with  regard  to  instruments  in  common, 
every-day  use  such  as  are  required  for  making  incisions,  exposing  vessels, 
etc.  Bistouries,  scissors,  forceps,  retractors,  inoculation  needles,  suture 
needles,  etc.,  must  all  be  sterilized  before  an  operation  either  in  the  hot  air 
sterilizer  at  180°  C.  or  by  boiling  in  water  for  10  minutes,  and  then  trans- 
ferring to  a  0*1  per  cent,  solution  of  oxycyanide  of  mercury  [or  2  per  cent, 
lysol].  When  the  operation  is  over  the  instruments  must  be  cleaned,  passed 
through  alcohol  and  dried  with  a  piece  of  soft  cloth. 

Sterile  absorbent  wool,  thread  and  silk  should  be  at  hand,  ready  for  use 
when  needed. 

Preparation  of  sterile  silk. — (a)  The  silk  may  be  sterilized  just  before  it  is  wanted 
by  boiling  it  for  15  minutes  in  3  per  cent,  carbolic  acid.  But  it  is  better  to  keep 
a  quantity  in  stock  prepared  by  one  or  other  of  the 
following  methods  : 

(6)  Cut  the  silk  into  lengths  of  about  12  inches  and 
wrap  three  or  four  lengths  in  two  or  three  pieces  of 
filter  paper.  Prepare  a  number  of  these  packets  of  silk 
and  heat  them  to  120°  C.  in  the  autoclave,  dry  them 
in  the  incubator  and  keep  them  in  a  well-stoppered 
bottle  in  a  box  with  a  tightly-fitting  lid.  Open  the 
packets  one  by  one  as  they  are  wanted.  Any  packet 
which  has  been  opened  and  not  used  must  be  thrown 
away. 

(c)  Place  the  reel  of  silk  and  a  little  water  in  a  small 
bottle.  Pass  the  end  of  the  silk  through  a  narrow 
piece  of  glass  tubing  which  should  perforate  the  cork 
of  the  bottle  and  be  plugged  with  wool  (fig.  132). 
Sterilize  in  the  autoclave.  When  the  silk  is  wanted  it 
is  only  necessary  to  pull  on  the  end  of  it  to  unreel 
it.  So  much  of  the  silk  thread  as  projected  before  it 
was  pulled  must  of  course  be  cut  off  and  thrown  away, 
keep  sterile  so  long  as  the  wool  plug  remains  in  position. 

A  solution  of  some  antiseptic  (e.g.  O'l  per  cent,  oxycyanide  of  mercury) 
[or  2  per  cent,  lysol]  and  some  sterile  water  must  also  be  at  hand. 


FIG.  132. — Bottle  for  storing 
sterile  silk. 

The  silk  in  the  bottle  will 


166 


EXPERIMENTAL  INOCULATIONS 


Preparation  of  sterile  water. — A  number  of  test-tubes  or  small  flasks  of  50-100  c.c. 
capacity  should  be  three-quarters  filled  with  water  and  sterilized  at  115°  C.  When 
wanted,  aspirate  the  water  out  of  the  tubes  or  flasks  with  a  Pasteur  pipette.  The 
contents  of  any  tube  or  flask  opened  and  not  used 'should  be  thrown  away  at  once. 

Water  may  also  be  sterilized  in  larger  quantities  in  flasks  :  but  it  is  better  to 
use  small  vessels  containing  only  sufficient  water  for  one  experiment.  All  risk  of 
contamination  is  then  avoided. 

A  number  of  sterile  glass  dishes,  glass  rods,  platinum  wires  and  Pasteur 
pipettes  must  also  be  ready. 

Lastly,  syringes  and  needles  are  necessary  with  which  to  inoculate  the 
virus  into  the  tissues. 

(i)  Inoculation  syringes. 

There  are  numerous  patterns  of  inoculation  syringes,  and  this  fact  in  itself 
shows  how  difficult  it  is  to  obtain  a  syringe  which  fulfils  all  the  conditions 
required  of  it.  A  satisfactory  syringe  should — 

1.  Lend  itself  to  sterilization  in  boiling  water  or  in  steam  under  pressure. 

2.  Have  perfectly  water-tight  joints  and  plunger,  and  be  so  constructed 
as  not  to  need  frequent  renewal  of  the  parts. 

3.  Be  accurately  graduated  on  the  glass  barrel  or  piston  rod. 

Pasteur  pipettes. — A  Pasteur  pipette  may  on  occasion  be  used  as  an  inoculation 
syringe.  And  for  this  purpose  the  drawn-out  part  is  made  short  and  slightly 
curved,  with  a  sharp  point  (fig.  133).  [The  opposite  end  is  plugged 
with  wool  and  the  pipette  sterilized  before  use.  A  small  bulb  may 
be  very  conveniently  blown  on  the  tube.  ]  The  liquid  to  be  inoculated 
is  sucked  up  into  the  pipette  and  the  pointed  end  pushed  through 
the  skin  [or,  when  required,  into  the  peritoneal  cavity],  and  the 
material  deposited  in  the  tissues  by  blowing  through  the  plugged  end. 
This  is  all  the  apparatus  that  is  necessary  in  a  large  number  of  cases. 

A.  Older  patterns. — Of  the  older  patterns  of  syringes  the  use 
of  which  is  now  being  given  up,  the  following  may  be  men- 
tioned : 

Pravaz's  syringe. — One  of  the  oldest  and  at  the  same  time  one  of 
the  best  from  the  point  of  view  of  the  security  of  its  joints  is  Pravaz's 
syringe.  But,  unfortunately,  the  joints  and  the  leather  plunger  will 
not  stand  the  temperature  of  boiling  water.  If  it  be  used  it  must 
be  disinfected  by  soaking  it  in  a  5  per  cent,  solution  of  carbolic  acid 
for  several  hours  and  subsequently  rinsing  well  in  sterile  water. 

Syringes  with  air  piston. — Koch,  Petri  and  others  eliminated  the 
plunger.  The  glass  cylinder  forming  the  body  of  the  syringe  was  fitted 
at  one  end  with  a  needle  and  at  the  other  with  an  india-rubber  ball : 
FIG.  133.—  by  squeezing  the  ball,  the  liquid  was  forced  out.  When  used  for 

Pasteur  pipette   injection  however  especially  into  tense  tissues,  the  liquid  either  cannot 

modified  for  m-    ,   J.  1,1  \ \        .1  -n    •        -/i   i  mi. 

oculations.          be  inoculated  or  runs  out  when  the  needle  is  withdrawn.    These  syringes 

are  not  now  in  use. 

Straus'  syringe. — By  substituting  compressed  elder  pith  for  the  leather  in  Pravaz's 
plunger,  Straus  obtained  a  syringe  which  stands  the  temperature  of  boiling  water  and 
of  the  autoclave  very  well.  The  plunger  can  be  changed  as  often  as  is  necessary, 
but  though  it  is  easily  done  it  takes  some  time  ;  moreover  it  has  to  be  done  fre- 
quently since  the  elder  pith  rapidly  loses  its  elasticity.  With  these  reservations 
the  syringe  is  a  good  one. 

To  renew  the  plunger. — Take  a  piece  of  elder  pith  with  a  regular  and  fine  grain, 
cut  off  the  outer,  fibrous  layer,  and  compress  it  between  the  fingers  so  as  to  flatten 
it  longitudinally  as  much  as  possible.  Then  cut  out  a  small  cylindrical  piece  to 
fit  the  barrel  of  the  syringe  tightly.  Perforate  its  centre  with  a  needle  heated  to 
redness  in  the  flame  and  fix  it  on  to  the  end  of  the  piston  rod.  Then  with  a  very 
fine  file  polish  its  sides  and  introduce  it  into  the  barrel.  By  soaking  in  water  for  a 
few  minutes  the  elder  pith  swells  and  the  plunger  becomes  water-tight.  The 
pith  can  also  be  compressed  at  will  by  means  of  the  screw  at  the  top  of  the  piston  rod. 


SYRINGES 


167 


In  Roux's  modification  the  glass  barrel  is  narrowed  below  and  ground  so  that 
the  needle  is  fitted  directly  on  to  it.  The  plunger  can  be  freely  withdrawn  or 
inserted  since  the  tube  is  merely  fitted  above  with  a  plug. 

Malassez's  syringe. — There  are  several  patterns  of  this  syringe.  The  only  ones 
which  can  be  recommended  are  those  in  which  the  plunger  consists  of  a  mixture  of 
india-rubber  and  asbestos,  or  of  "fibre," 
a  combination  of  cellulose  and  rubber. 
The  lower  end  is  narrowed  and  ground  and 
the  needle  fitted  on  to  it  by  means  of  a 
"'  fibre  "  washer. 

Metal  plunger  syringes. — In  these  forms 
the  elastic  plunger  is  replaced  by  a  rigid 
piston  consisting  of  an  accurately  calibrated 
metal  rod,  the  body  itself  being  an  hollow 
metal  cylinder.  These  syringes  soon  de- 
teriorate and  are  inconvenient  in  that  the 
liquid  within  them  is  not  visible. 

B.  Patterns  recommended  for  use. 
Roux's  syringe  (fig.  134). — For  serum- 
therapeutic  inoculations,  Roux  devised 
a  syringe  of  20  c.c.  capacity  with  a 
plunger  made  of  some  rubber  pre- 
paration. The  needle  is  connected  to 
the  nozzle  of  the  barrel  by  a  piece  of 
rubber  tubing  about  10  cm.  long.  This 
arrangement  allows  the  injection  to  be 
forced  into  the  tissues  without  the 
risk  of  detaching  the  needle.  Before 
sterilizing  the  syringe  be  careful  to 
loosen  the  upper  socket  to  leave  the 
glass  barrel  room  to  expand,  and  so 
prevent  it  being  cracked. 

Debove's  syringe  (fig.  135). — In  the 
author's  opinion  Debove's  syringe  is 
better  than  Roux's.  It  is  both  easy 
to  manipulate  and  easy  to  sterilize :  it 
is  solid  and  perfectly  water-tight  and 
can  be  used  for  all  sorts  of  inoculations. 

The  syringe  consists  of  a  glass  tube 
held   between  two  metal    sockets  by 
means  of  a  movable  metal  armature 
which  is  entirely  distinct  and  controlled  by  a  lever.     The  barrel  is  accurately 
calibrated  and  the  syringe  is  made  water-tight  with  asbestos  washers. 

The  lower  metal  socket  has  a  conical  extension  on  to  which  the  needle 
fits  either  directly  or  through  an  india-rubber  connexion. 

The  plunger  consists  of  asbestos  rings  held  between  two  metal  discs.  The 
piston  rod  carries  a  screw  which  allows  the  pressure  on  the  asbestos  rings 
forming  the  plunger  to  be  varied  so  that  the  play  of  the  plunger  can  be 
regulated. 

The  syringe  is  easily  taken  to  pieces  by  raising  the  lever ;  this  relaxes 
the  lateral  stays  which  can  then  be  disconnected  and  the  sockets  taken  off. 

All  parts  of  the  syringe  are  made  to  a  standard  pattern,  so  that  broken 
parts  can  be  replaced  without  sending  it  to  the  maker  to  be  repaired.  The 
syringe  is  made  in  several  sizes  to  hold  from  2-100  c.c.,  those  ordinarily  in 
use  being  of  2,  10  and  20  grams'  capacity. 


FIG.  134. — Roux's 
syringe. 


FIG.  135. — Debove's 
syringe. 


168 


EXPERIMENTAL  INOCULATIONS 


Method  of  sterilizing  Debove's  syringe. — Withdraw  the  plunger  as  far  as  possible, 
raise  the  lever  to  relax  the  spring  and  allow  expansion  of  the  glass  cylinder.  Place 
the  syringe  and  needle  in  a  vessel  of  cold  water  and  heat  to  boiling  for  15  or  20 
minutes.  Let  the  syringe  cool ;  then  take  it  out  of  the  water  with  a  pair  of  sterile 
forceps,  let  the  water  above  the  plunger  run  out,  lower  the  lever  and  fit  the  needle 
on  its  socket. 

(When  the  syringe  has  been  used  for  an  injection.,  rinse  it  out  in  cold  water  to 

wash  out  all  albuminoid  matter — which 
would  coagulate  on  boiling — and  boil  it 
in  the  same  water,  so  that  both  the  latter 
and  the  syringe  are  sterilized  at  the  same 
time. ) 

The  syringe  may  be  sterilized  in  the 
autoclave  if  preferred  :  it  is  prepared  as 
above  and  then  heated  to  115°  C.  for  a 
quarter  of  an  hour.  In  most  cases,  boiling 
is  sufficient  to  completely  sterilize  it,  but 
when  it  has  been  used  for  inoculating  cul- 
tures of  spore- bearing  bacilli,  such  as  B. 
tetani,  B.  maligni  oedematis,  etc.,  it  should 
be  autoclaved. 

Syringes  with  glass  pistons. — Malassez 
has  had  a  syringe  made  by  Luer  which, 
is  entirely  of  glass.  The  piston  itself  con- 
sists of  a  calibrated  glass  rod.  Numerous 
forms  of  syringes  based  on  this  pattern 
can  now  be  bought  at  a  low  price. 

These  syringes  are  easily  sterilized  quite  water-tight  and  are  excellent  in 

every  way,  particularly  for  small  volumes  (1-2  c.c.). 

Apparatus  for  injecting  large  quantities  of  fluid.— In  immunizing  animals, 
with  toxins  when  large  quantities  of  filtered  cultures  are  inoculated,  syringes 
are  not  large  enough,  and  moreover  the  fluid  cannot  be  injected  sufficiently 
slowly.  In  these  cases  the  following  arrangement 
is  useful  (fig.  137). 

The  liquid  to  be  inoculated  is  poured  into  a  tall 
glass  vessel  graduated  on  the  glass  from  above 
downwards  and  closed  with  an  india-rubber  plug 
which  is  perforated  by  two  glass  tubes,  one  of 
which,  reaching  to  the  bottom  of  the  vessel,  has  a 
needle  attached  to  its  upper  end  with  india-rubber 
tubing.  The  other  tube  passes  a  few  centimetres 
below  the  stopper,  is  plugged  with  wool,  and 
through  it  the  air  in  the  vessel  can  be  compressed. 
When  this  is  done  the  liquid  flows  out  of  the 
needle,  and  the  rate  of  flow  can  be  regulated  at 
will. 

The  apparatus  is  sterilized  in  the  autoclave 
and  then  the  liquid  to  be  inoculated  aspirated 
into  the  vessel. 


FIG.  136. — Another  reliable  form  of  syringe 
("  The  Record  "). 


FIG.   137. — Apparatus  for  inocu- 
lating large  volumes  of  liquids. 


(ii)  Needles. 

Steel  needles  are  very  generally  used  for  inocu- 
lations. The  disadvantage  of  steel  is  that  it  so 
readily  rusts  with  the  result  that  the  lumen  of  the  needle  soon  becomes  ob- 
structed. This  difficulty  is  overcome  by  carefully  washing  the  needles  after 
use,  and  keeping  them — after  they  have  been  sterilized  by  boiling — in  a  small 


NEEDLES 

bottle  filled  with  absolute  alcohol  [to  which  a  few  lumps  of  calcium  chloride 
may  with  advantage  be  added]  or  in  a  3  per  cent,  solution  of  sodium  borate. 

On  account  of  the  difficulty  of  preventing  steel  from  rusting  platinum-iridium 
needles  are  gradually  replacing  steel.  Platinum-iridium  does  not  rust  and 
the  needles  can  be  heated  to  redness.  On  the  other  hand  they  are  expensive 
and  delicate  and  as  they  are  but  little  stronger  than  needles  made  of  pure 
platinum,  it  is  on  the  whole  better  to  use  steel  especially  when  a  thick  skin 
has  to  be  penetrated  as  is  generally  the  case  in  animal  inoculation. 

A  selection  of  needles  of  different  calibre  and  of  different  lengths  ought  to- 
be  kept  in  the  laboratory. 

2.  Preparation  of  the  material  for  inoculation. 

The  material  to  be  inoculated  may  be  either  a  solid  or  a  liquid.  The 
procedure  will  be  different  in  the  two  cases. 

(i)  Of  fluids. 

Broth  cultures  are  the  commonest  fluids  inoculated  but  other  fluids,  such 
as  blood,  serum,  pleural  and  peritoneal  exudates,  have  also  to  be  inoculated 
at  times. 

(a)  Cultures. — Every  culture  should  be  examined  microscopically  before  being 
inoculated,  to  test  its  purity. 

When  ready  to  perform  the  inoculation  remove  a  little  of  the  culture  with 
a  Pasteur  pipette  and  transfer  it  to  a  sterile  watch-glass  and  cover  the  latter 
again  with  the  paper  in  which  it  was  sterilized.  Aspirate  the  culture  into  a 
sterile  syringe  through  the  needle  either  by  puncturing  the  paper  with  the 
needle  or  by  slightly  raising  the  paper  and  passing  the  needle  beneath  it. 
Hold  the  syringe  with  the  needle  pointing  upwards  and  gently  press  the 
plunger  to  expel  any  air  which  may  have  been  drawn  into  the  syringe,  taking 
care  to  hold  the  piece  of  sterile  paper  which  covered  the  watch-glass  along- 
side the  needle  to  catch  any  drops  of  culture  which  may  inadvertently  be 
driven  out.  Burn  the  paper  and  dip  the  watch-glass  into  a  vessel  of  boiling 
water  to  sterilize  it. 

(b]  Exudates. — Blood  and  serous  exudates  must  be  collected  in  the  manner 
to  be  described  in  Chaps.  XL  and  XII.     With  a  pipette  transfer  the  amount 
required  for  inoculation  to  a  sterile  watch-glass  and  proceed  as  above.     It 
is  very  difficult  to  inject  blood  directly  because  it  so  readily  coagulates  and 
blocks  the  needle.     If  the  virus  pass  into  the  serum,  the  blood  should  be 
allowed  to  clot  and  the  serum  used  for  inoculation.     On  the  other  hand  if 
the  virus  be  retained  in  the  clot  this  should  be  dealt  with  as  though  it  were 
a  solid  tissue  (vide  infra}. 

To  facilitate  the  inoculation  of  whole  blood  it  is  occasionally  necessary  to  have 
resort  to  the  anti-coagulating  action  of  sodium  citrate.     The  blood  is  collected  in  a 
sterile  vessel  containing  a  little  of  the  following  solution  also  sterilized : 
Water,  -  1000  c.c. 

Sodium  chloride,      -  8  grams. 

Sodium  citrate,        -  -         15       ,, 

Use  two  to  four  volumes  of  the  citrate  solution  to  one  volume  of  blood.  Mix 
thoroughly  and  inoculate  without  delay. 

(ii)  Of  solid  substances. 

(a)  Solid  substances. — Fragments  of  internal  organs,  splinters,  etc.,  maybe 
inserted  directly  into  the  tissues  of  the  animal.  After  making  a  small  incision 
separate  the  cellular  tissue  with  a  director  and  introduce  the  material  into 
the  pocket  so  formed,  suture  the  wound  and  cover  it  with  collodion.  Material 
may  be  similarly  introduced  into  the  peritoneal  cavity,  muscles,  etc. 


170  EXPERIMENTAL  INOCULATIONS 

(b)  Cultures  on  solid  media. — A  small  incision  may  be  made  in  the  skin 
and  then  a  wire,  charged  with  the  micro-organisms  by  scraping  the  surface 
of  the  medium,  rubbed  into  the  tissues.     But  it  is  more  usual  to  make  an 
emulsion  by  rubbing  up  some  of  the  material  in  sterile  water  or  broth  and 
then  to  inoculate  the  emulsion  with  a  syringe. 

Scrape  the  surface  of  the  medium  with  a  stout  platinum  wire  and  transfer 
the  growth  to  a  sterile  watch-glass  containing  a  little  sterile  water  and  stir 
it  about  until  an  homogeneous  emulsion  is  obtained.  If  the  culture  be  difficult 
to  break  up — as,  for  example,  a  growth  of  the  tubercle  bacillus — and  does  not 
mix  with  water,  it  should  be  ground  up  as  described  below  (d). 

(c)  Pus. — As  a  rule,  pus  is  too  thick  to  be  inoculated  undiluted.     Transfer 
a  few  drops  of  the  pus  with  a  pipette  to  a  sterile  watch-glass,  add  a  little 
sterile  normal  saline  solution  (O8  per  cent,  aqueous  solution  of  sodium  chloride) 
or  broth  and  mix  them  thoroughly  with  the  end  of  the  pipette. 

(d)  Fragments   of  organs. — For  the   method   of  collecting  fragments  of 
internal  organs,  portions  of  the  central  nervous  system,  etc.,  see  Chap.  XI. 

Transfer  the  material  to  a  sterile  watch-glass  and  break  it  up  with  a  sterile 
glass  rod.  When  the  tissue  is  reduced  to  a  fine  paste  add  a  little  sterile  normal 
saline  solution  drop  by  drop  from  a  pipette,  and  mix  until  a  quite  homo- 
geneous suspension  is  obtained. 

It  is  often  necessary  to  filter  the  suspension  through  a  small  piece  of  pre- 
viously sterilized  fine  muslin  to  get  rid  of  little  lumps.  This  precaution  is 
very  necessary  when  the  emulsion  has  to  be  inoculated  intra-venously,  in 
order  to  avoid  the  formation  of  an  embolus. 

When  a  very  tough  material  has  to  be  dealt  with  such  as  tuberculous  or 
leprous  nodules,  it  should  be  cut  up  into  quite  small  fragments  with  sterile 
scissors,  ground  up  in  a  sterile  mortar  with  some  fine  sterile  sand  x  (p.  155) 
and  a  fluid  emulsion  made  by  adding  sterile  normal  saline  solution  drop  by 
drop  :  the  emulsion  is  then  filtered  through  fine,  sterile  muslin  before  being 
inoculated. 

When  a  larger  quantity  of  material  has  to  be  emulsified,  BorrePs  broyeur 
will  be  found  useful.  With  this  machine  fine  powders  or  emulsions  can  be 
obtained  without  contaminating  the  material  and  without  exposing  the 
operator  to  the  inhalation  of  dust  containing  pathogenic  organisms. 

3.  Technique  of  inoculation. 

General  rules. — Before  inoculating  an  animal  shave  the  hair  and  cleanse 
the  skin  of  the  part. 

The  hair  may  be  cut  very  short  with  a  pair  of  curved  scissors  but  it  is  better  to 
shave  the  skin.  For  delicate  inoculations  it  is  preferable  to  epilate  the  hairs  with 
one  of  the  following  solutions : 

1.  Recently  slaked  lime,       -  2  parts. 
Water,  -  3     „ 

Pass  a  stream  of  hydrogen  sulphide  through  the  emulsion,  shaking  it  frequently, 
until  it  is  saturated.  Apply  the  paste  to  the  part  to  be  epilated  and  after  a  few 
minutes  wash  it  off  with  water  and  a  nail  brush. 

2.  Sodium  sulphide,     -  3  parts. 
Powdered  quicklime,        -                                                                       10       ,, 
Starch,                                                                                           -         10       „ 

Mix  into  a  powder.  When  required  for  use,  add  sufficient  water  to  a  little  of  the 
powder  to  form  a  soft  paste  and  apply  it  to  the  skin.  After  3  or  4  minutes'  applica- 
tion the  hair  is  removed. 

I1  Sand  was  used  as  a  triturating  agent  by  Cobbett  in  his  earlier  experiments  on  tuber- 
culosis but  was  almost  immediately  given  up.  A  little  patience  is  all  that  is  required  to 
grind  up  even  a  tough  tuberculous  lesion.] 


SUB-CUTANEOUS  INOCULATION  171 

In  a  large  number  of  cases  it  is  only  necessary  after  cutting  the  hair,  to  rub 
the  skin  with  a  [2  per  cent,  solution  of  lysol  or  a]  O'l  per  cent,  solution  of  oxy- 
cyanide  to  render  it  aseptic.  But  for  more  perfect  asepsis  the  skin  should 
be  scrubbed  with  soap  and  a  nail  brush,  washed  with  an  antiseptic  solution, 
rinsed  with  alcohol  and  wiped  with  sterile  filter  paper. 

(i)   Intra-dermal  inoculation. 

1.  Shave  and  cleanse  the  part  but  use  no  antiseptics. 

2.  Scarify  the  skin  very  superficially  with  a  bistoury,  or  pick  up  the  skin 
in  the  fingers  and  shave  off  the  epidermis  with  the  blade  of  a  sharp  knife. 

3.  Bub  the  material  to  be  inoculated  into  the  prepared  part  with  a  sterile 
glass  rod  or  a  piece  of  sterile  wool  held  in  a  pair  of  forceps. 

In  some  cases,  it  is  sufficient  merely  to  rub  the  skin  briskly  with  a  sponge  soaked 
in  the  material  without  scarifying  or  scraping  the  surface. 

As  a  rule,  the  material  should  be  inoculated  into  some  part  of  the  skin  of 
the  body  which  the  animal  cannot  reach,  such  as  the  dorsal  surface  of  the 
ear,  the  skin  of  the  back,  or  the  root  of  the  tail.  This  rule  applies  to  the 
inoculation  of  all  species  of  laboratory  animals.  For  some  viruses  there  are 
special  sites  used  for  inoculation,  the  eyelids  or  eyebrows  in  syphilis,  for 
example. 

(ii)  Inoculation  on  the  surface  of  mucous  membranes. 

Abrade  the  surface  of  the  mucous  membrane,  as  in  the  preceding  case,  by 
scraping  it  with  the  blade  of  a  knife,  and  spread  the  virus  over  the  surface 
thus  abraded.  Sometimes  it  is  better,  before  inoculating  the  mucous  mem- 
brane, to  cauterize  it  with  a  moderately  hot  iron  or  platinum  rod  so  as  to 
produce  a  superficial  slough. 

(iii)  Sub-cutaneous  inoculation. 

A.  Of  a  liquid. — 1.  Shave  and  cleanse  the  skin. 

2.  Pick  up  a  fold  of  skin  between  the  thumb  and  index  finger  of  the 
left  hand,  insert  the  needle  at  the  base  of  the  fold,  inject  the  fluid  and  with- 
draw the  needle.  Care  must  be  taken  to  see  that  the  fluid  does  not  find  its 
way  out  again  through  the  needle  puncture  and  that  the  injection  is  not 
made  into  the  muscles.  [By  lightly  pressing  the  fold  in  which  the  puncture 
has  been  made  between  the  finger  and  thumb  and  twisting  it  gently,  exuda- 
tion through  the  puncture  can  almost  always  be  prevented.] 

B.  Of  a  solid. — 1.  Shave  and  cleanse  the  part. 

2.  Make  a  small  incision  through  the  skin  with  a  bistoury,  separate  the 
sub-cutaneous  tissue  with  a  director  over  a  sufficient  area  and  then  introduce 
the  material  to  be  inoculated  into  the  pocket  with  a  pair  of  sterile  forceps. 

3.  Put  a  stitch  or  two  into  the  incision  and  cover  it  with  a  little  collodion. 
Note. — As   has  already   been   stated  above,  inoculations  are  most  satisfactorily 

made  into  some  part  of  the  body  that  the  animal  cannot  reach  ;   they  are  however 
often  made  beneath  the  skin  of  the  abdomen  or  thigh. 

When  the  material  to  be  inoculated  is  solid,  some  part  of  the  body  where  the  skin 
is  very  loose  should  be  chosen,  as  for  instance,  the  flanks  or  the  groin. 

(iv)  Inoculation  into  lymphatic  spaces. 

In  the  frog,  inoculations  are  frequently  made  into  the  sub-cutaneous 
connective  tissue  which  consists  of  large  inter-communicating  lymphatic 
sacs. 

A.  Dorsal  sac. — The  dorsal  sac  is  situated  over  the  posterior  part  of  the 
back.  The  animal's  hind  limbs  are  wrapped  in  a  cloth  so  that  it  cannot 
move,  and  by  compressing  the  sides  of  the  back  with  the  fingers  the  sac  is 


172 


EXPERIMENTAL  INOCULATIONS 


made  to  stand  out.     A  fine  needle  is  introduced  obliquely  into  the  sac  from 
above  downwards  and  the  material  injected. 

B.  The  posterior  limb  sacs. — Introduce  a  needle  obliquely  from  above 
downwards  under  the  skin  below  the  femoro-tibial  joint  and  inject  the 
material. 

(v)  Intra-muscular  inoculation. 

1.  Shave  and  cleanse  the  part. 

2.  Push  the  needle  deeply  into  the  muscles,  inject  the  material  and  with- 
draw the  needle. 

Inoculations  are  made  for  preference  into  the  muscles  of  the  thigh  in  the 
mammalia,  and  into  the  pectoral  muscles  of  birds. 

(vi)  Intra-venous  inoculation. 

Whenever  possible  intra-venous  inoculation  should  be  made  into  a  superficial 
vein.  The  needle  may  be  passed  through  the  skin  directly  into  the  vein 
without  first  exposing  the  latter.  Intra-venous  inoculation  cannot  be  effected 
in  the  case  of  very  small  animals  such  as  mice. 

A.  Rabbits. — 1.  One  of  the  dorsal  veins  of  the  ear,  and,  for  preference, 
the  external  marginal  vein  should  be  selected.  Avoid  the  median  veins, 
because,  being  embedded  in  a  lax  cellular  tissue,  they  are  liable  to  slip  away 
from  under  the  needle. 

2.  Cut  the  hair  over  the  vein  with  a  pair  of  curved  scissors  [or  better,  shave 


FIG.  138. — Pressure  forceps  for  the  ear  vein. 

it],  and  cleanse  the  skin.     The  rabbit  is  placed  on  the  operator's  knee  and, 
if  necessary,  held  by  an  assistant. 

3.  Take  hold  of  the  margin  of  the  ear  between  the  index  finger  and  thumb 
of  the  left  hand  so  as  to  extend  it.  Put  a  pair  of  pressure  forceps  on  the 
vein  at  the  base  of  the  ear  so  as  to  make  the  vein  prominent  (fig.  138). 

By  rubbing  the  skin  with  a  sponge 
soaked  in  warm  carbolic  water  the  vein 
can  be  rendered  more  distinct. 

4.  Hold  the  needle  very  obliquely,  al- 
most parallel  to  the  vessel  and  pierce  it 
in    the    direction    of    the    blood-stream 
(fig.  139). 

5.  When  the  needle  is  in  the  vein,  take 
off  the  forceps  and  inject  the  fluid  slowly. 
It  is  a  good  plan  to  apply  the  forceps  higher 
up  on  the  needle  itself,  so  as  to  hold  it 
in  the  skin.     If  the  needle  has  missed  the 

^  vein,    the    injection    will    cause    a   sub- 

/  M^L-^^_ ^  cutaneous    swelling,    and    the    operation 

S  must  be  begun  again  lower  down. 

After  the  fluid  is  injected  withdraw  the 
needle,  and  if  the  vessel  bleed  leave  the 
forceps  on  the  bleeding  point  for  a  few  minutes,  [or  pass  the  vein  between 
the  finger  and  thumb  moving  them  against  the  blood-stream]. 

B.  Guinea-pigs. — The  superficial  veins  are  not  large  enough  in  the  guinea- 


FlG.  139. — Inoculating  into  the  ear  vein 
of  a  rabbit. 


INTRA- VENOUS  INOCULATION  173 

pig  for  intra-venous  inoculation,  and  recourse  must  be  had  to  the  external 
jugular.  This  vein  is  superficially  situated,  lying  beneath  the  skin  the  sub- 
cutaneous muscles  and  some  cellular  tissue,  and  follows  a  line  from  the  angle 
of  the  jaw  to  a  point  mid-way  between  the  shoulder  and  the  sternum. 

1.  Fix  the  guinea-pig  on  its  back,  with  its  head  extended.     Shave  and 
cleanse  the  part. 

2.  Make  an  incision  through  the  skin  and  sub-cutaneous  muscles  in  the 
middle  of  the  line  described  above,  tear  through  the  cellular  tissue  with  a 
director,  and  the  vein  will  be  exposed  lying  to  the  outer  side  of  the  incision. 

3.  Pass  the  needle  obliquely  into  the  vein  (it  is  very  convenient  to  have  a 
needle  with  the  lower  end  bent  at  a  right  angle)  inject  the  fluid  and  with- 
draw the  needle. 

4.  Cleanse  the  wound  with  a  sponge  soaked  in  carbolic  water  and  make 
quite  sure  that  there  is  no  bleeding  from  the  prick  in  the  vein.     Put  two  or 
three  stitches  in  the  skin  and  cover  the  incision  with  collodion. 

C.  Dogs. — For  intra-venous  inoculation  in  dogs  the  external  vein  of  the 
hind  limb — the  small  saphenous — should  be  selected. 

1.  Muzzle  the  animal,  and  get  an  assistant  to  hold  it. 

2.  Shave  the  skin  on  the  outer  side  of  the  limb  where  the  calf  muscles 
are  inserted  into  the  Tendo  achillis.     Compress  the  limb  above,  and  rub  the 
shaved  part  with  a  sponge  soaked  in  carbolic  water.     The  small  saphenous 
vein  will  thus  be  made  to  stand  out  and  is  easily  accessible  at  the  upper 
part  of  the  Tendo  achillis. 

3.  Avoid,  if  possible,  having  to  expose  the  vein,  and  in  performing  the 
inoculation  pierce  the  skin  and  the  vein  at  one  and  the  same  time. 

D.  Horses  and  Cattle. — Locate  the  jugular  vein  and  render  it  prominent 
as  described  on  p.  49.     Make  the  injection  with  the  usual  precautions. 

E.  Birds. — Birds  are  best  inoculated  intra-venously  in  the  axillary  vein. 

1.  Fasten  the  bird  down,  and  let  an  assistant  extend  the  wing,  and  at 
the  same  time  compress  the  base.     Pluck  the  down  from  the  inner  surface 
of  the  root  of  the  wing,  and  rub  the  part  with  a  sponge  soaked  in  carbolic 
water. 

2.  When  the  vein  has  swelled,  inoculate  the  material. 

(vii)  Arterial  inoculation. 

In  mammals  for  purposes  of  arterial  inoculation  the  femoral  or  carotid 
artery  is  chosen. 

A.  Femoral  artery. — The  femoral  artery  takes  the  same  course  in  animals 
as  it  does  in  man.  In  the  fold  of  the  groin,  the  vein  is  on  the  inside,  the 
artery  next  and  the  crural  nerve  on  the  outer  side.  The  artery  takes  a  line 
from  the  middle  of  the  fold  of  the  groin  to  the  inner  side  of  the  knee. 

1.  Fix  the  dog  on  its  back.     Rotate  the  leg  outwards  and   extend   it. 
Shave  and  cleanse  the  part. 

2.  Determine  the  exact  position  of  the  artery  by  finding  the  pulse  near 
the  middle  of  the  fold  of  the  groin,  and  make  an  incision  through  the  skin 
and  sub-cutaneous  tissue,   a  few  centimetres  long,  along  the  line    of   the 
vessel. 

3.  Divide  the  aponeurosis  on  a  director  and  the  sheath  of  the  vessels 
and  nerve  will  be  exposed. 

4.  Having  found  the  artery  prick  it  very  obliquely,  inject  the  material 
and  withdraw  the  needle. 

5.  Put  a  few  stitches  in  the  skin  and  paint  the  wound  over  with  collodion. 


174  EXPERIMENTAL  INOCULATIONS 

B.  Carotid. — In  all  mammals,  the  carotid  artery  lies  in  close  relation  to- 
the  trachea  in  the  middle  of  the  neck,  being  contained  in  a  sheath  common 
to  it,  the  internal  jugular,  the  pneumogastric  and  the  great  sympathetic. 

1.  With  the  animal  lying  on  its  back  and  its  head  extended  shave  the  skin 
of  the  middle  of  the  neck  and  wash  it  with  an  antiseptic. 

2.  Make  a  longitudinal  incision,  a  few  centimetres  long,  in  the  middle  line, 
in  front  of  the  trachea. 

3.  Divide  the  aponeurosis  connecting  the  two  sterno-mastoids  on  a  director. 

4.  Separate  the  cellular  tissue  along  the  trachea  with  the  rounded  end  of 
a  probe  and  then,  by  pulling  the  sterno-mastoid  outwards,  the  sheath  of  the 
vessel  will  come  into  view. 

5.  Open  the  sheath  with  a  pair  of  forceps  and  a  director.     The  artery  will 
be  recognized  from  the  vein  by  its  larger  size.     Make  the  injection  as  described 
above. 

(viii)  Intra-peritoneal  inoculation. 

A.  Of  a  fluid. — Every  precaution  should  be  taken  not  to  perforate  the  gut. 

1.  Fix  the  animal  firmly  on  its  back.     [An  assistant  can  hold  it  equally 
well.]     Shave  and  disinfect  a  few  square  centimetres  of  the  skin  of  the 
abdomen. 

2.  Pick  up  the  whole  thickness  of  the  abdominal  wall  between  the  thumb 
and  index  finger  of  the  left  hand. 

3.  Insert  the  needle  of  the  syringe  into  the  base  of  the  fold  in  such  a  way 
that  the  point  is  directed  upwards,  withdraw  it  a  little,  and  then,  altering 
the  direction  of  the  point,  pass  it  into  the  cavity  of  the  abdomen.     Inject 
the  material  and  withdraw  the  needle. 

The  following  method  affords  greater  security.  A  curved 
needle  is  used,  in  which  the  opening  is  situated  in  the 
centre  of  the  concavity  of  the  arc  (fig.  140). 

Insert  the  needle    through   the  whole  thickness  of  the 
abdominal  wall,  including  the  peritoneum,  and  bring  the 
%n^o^noceulationtra"    Point  to  the  surface ;    the  needle -opening  is  now  within 
the  peritonea]  cavity,  and  the  material  is  injected.     The 

point  of  the  needle  being  outside  all  the  time  that  the  injection  is  being    made  no 
injury  can  be  done  by  it  to  the  gut. 

B.  Of  a  solid. — 1.  The  animal  is  fixed  on  its  back,  and  the  skin  of  the 
part  shaved  and  cleansed. 

2.  Make  an  incision  in  the  median  line,  the  length  of  the  incision  varying 
with  the  size  of  the  substance  to  be  inoculated. 

3.  Cut  through  the  aponeurosis,  using  a  director  to  avoid  injuring  the 
intestine. 

4.  Take  hold  of  the  aponeurosis  on  each  side  with  pressure  forceps,  and 
hold  it  up  as  much  as  possible  to  prevent  the  intestines  protruding.     Intro- 
duce the  material  to  be  inoculated  into  the  wound  and  push  it  well  under  the 
muscular  layer  into  the  peritoneal  cavity. 

5.  Sew  up  the  aponeurosis  with  silk,  stitch  the  skin  and  cover  the  incision 
with  a  layer  of  collodion. 

Note. — The  most  careful  asepsis  is  necessary  in  performing  intra-peritoneal 
inoculations.  These  inoculations  should  always  be  done  with  pure  cultures  or 
with  material  which  can  be  obtained  free  from  contaminating  organisms  for,  if 
sputum,  excreta,  etc.,  are  injected  into  the  peritoneal  cavity  the  animal  will  very 
quickly  die  of  peritonitis.  [But  see  Chap.  XVIII.  Sect.  IV.] 

C.  Collodion  sacs. — In  their  researches  on  cholera  toxin,  Metchnikoff,  Roux 
and  Salimbeni  devised  a  method  of  growing  organisms  in  small  closed  collodion 
sacs  in  the  peritoneal  cavities  of  animals. 


COLLODION  SACS  175 

By  this  method,  organisms  can  be  cultivated  in  the  body,  and  at  the  same 
time  be  protected  from  phagocytes.  The  thin  walls  of  the  collodion  sacs, 
while  allowing  osmotic  changes  to  take  place  which  alter  the  composition 
of  the  medium  in  the  sacs  and  permit  the  toxins  secreted  by  the  organisms  to- 
diffuse  into  the  tissues  of  the  animal,  prevent  cells  (micro-organisms  and 
phagocytes)  passing  through.  This  method  has  many  applications  in  bac- 
teriological work. 

All  the  soluble  products  of  micro-organic  metabolism  dialyse  more  or  less 
through  the  walls  of  collodion  sacs,  but  they  do  not  pass  through  in  toto.  The 
collodion  membrane  does  not  act  as  a  perfect  filter,  so  that  some  toxins  pass  through 
only  with  difficulty  and  very  slowly  (Rodet  and  Guechoff).  The  immunizing 
substances  seem  to  pass  through  first  (Crendiropoulo  and  Ruffer). 

(a)  Method  of  preparing  collodion  sacs. — Have  ready  (1)  a  wide-mouthed 
bottle  containing  collodion,  free  from  castor  oil,  and  of  a  medium  syrupy 
consistence,  (2)  some  small  glass  tubes  of  5-6  mm.  internal  diameter,  (3)  some 
small  conical  india-rubber  plugs,  (4)  a  test-tube  carefully  calibrated  and  not 
enlarged  at  its  lower  part,  (5)  some  silk  thread. 

Collodion  containing  castor  oil  [collodium  flexile  B.P.  ]  may  be  used  instead  of 
ordinary  collodion  but  such  sacs  are  not  so  transparent,  though  they  are  more 
elastic  and  stronger  than  the  others. 

1.  Rotate  the  lower  end  of  the  test-tube  regularly  on  the  sloping  surface 
of  the  collodion  and  prolong  the  contact  according  to  the  thickness  which 
the  layer  of  collodion  is  to  have. 

2.  Remove  the  tube  from  the  collodion  and  continue  turning  it  between 
the  fingers  for  about  a  minute.     Then  let  the  layer  of  collodion  dry  for  a 
few  minutes  until  it  is  of  a  semi-liquid  consistence. 

3.  With  a  scalpel  cut  round  the  layer  of  collodion  near  its  upper  end  and 
separate  the  sac  from  the  tube,  thus :  Free  the  upper  end  of  the  sac  with  the 
thumb  nail,  turn  it  back  like  a  glove  finger  and  gradually  peel  it  off.     This 
must  be  done  slowly.     [Dipping  in  methylated  spirit  softens  the  collodion 
and  makes  it  strip  easily.] 

4.  Distend  the  sac  by  blowing  into  it.     A  sac  may  be  made  to  hold  from  one 
to  several  cubic  centimetres  according  to  the  particular  requirements  of  the 
experiment. 

5.  Fit  a  piece  of  small  glass  tubing  into  the  open  end  of  the  sac,  fasten  it 
on  with  several  turns  of  silk  thread  and  cover  the  silk  with  a  little  collodion. 
Fill  the  sac  with  water  and  suspend  it  by  a  thread  in  a  bottle  or  test-tube 
containing  a  little  water.     Plug  the  vessel  with  wool.     Place  the  india-rubber 
plugs  in  a  flask  plugged  with  wool  and  containing  a  little  water.     Sterilize 
at  115°  C.  in  the  autoclave. 

All  these  manipulations  are  delicate  and  take  a  long  time,  and  it  will  often  be 
advantageous  to  use  the  collodion  sacs  which  can  now  be  bought  in  the  shops. 

6.  Take  each  sac  out  of  the  bottle  with  a  pair  of  sterile  forceps  and 
transfer  it  to  a  sterile  dish  covered  with  paper.     Suck  up  the  water  out  of 
the  sac  with  a  pipette  and  replace  it  with  broth  sown  with  the  organism  under 
investigation. 

7.  Close  the  opening  of  the  tube  with  an  india-rubber  plug  picked  up  with 
sterile  forceps,  and  cut  it  off  short  close  to  the  tube  with  a  sterile  scalpel. 
Dehydrate  the  plug  in  absolute  alcohol  and  cover  it  with  several  layers  of 
collodion. 

Notes. — (i)  Bertarelli  has  recommended  a  method  which  considerably  simplifies 
that  just  described.  In  Bertarelli' s  method,  the  free  end  of  the  piece  of  glass  tubing 
to  which  the  sac  is  affixed  is  drawn  out  beforehand  so  that  the  free  end  is  fine  and 


176 


EXPERIMENTAL  INOCULATIONS 


conical.  After  the  sac  has  been  sterilized  the  water  is  withdrawn  with  a  syringe 
and  the  culture  introduced.  The  pointed  end  is  then  sealed  off  in  a  small  flame. 
Bertarelli  further  suggests  using  a  solution  of  collodion  in  ether  of  the  same 
consistence  as  is  used  for  embedding,  in  place  of  collodion. 

(ii)  Phisalix  has  introduced  a  modification  of  the  method  of  preparation  by 
which  much  stronger  sacs  can  be  made,  and  the  risk  of  breakage  in  the  abdominal 
•cavity  thereby  reduced. 

A  collodion  sac  is  prepared  as  above  (Stages  1  to  4)  and  slipped 
on  to  a  guide  consisting  of  a  perforated  glass  ampoule  (fig. 
141,  A).  The  sac  which  now  covers  the  guide  is  fastened  to  the 
neck  of  the  ampoule  with  a  few  turns  of  silk  thread,  and  this 
is  covered  with  a  layer  of  collodion. 

The  sac  is  then  sterilized  in  the  autoclave  in  the  ordinary 
way.  After  the  sac  has  been  filled  with  the  culture  the  neck 
of  the  ampoule  is  sealed  off  in  the  flame  (fig.  141,  B). 

\       /^^\         (**i)  Grorsline    noted  that  the  principal    difficulty  in    making 
collodion  sacs  is  the  separation  of   the   sac  from  the  tube  on 
which  it  is  moulded.     He   overcomes  this  difficulty   by   using 
3D  ;CD          ^  W:]     test- tubes  perforated  with  a  small  hole  in  the  bottom.     In  making 
a  sac  the  hole  is  first  obliterated  by  gently  touching  the  bottom 
O  D          &&:      °f  the  tu^e  on  *ke  collodion.     After  the  sac  has  then  been  made 
in  the  ordinary  way  and  dried,  the  tube  is  filled  with  water. 
By  blowing  down  the  open  end  of  the  tube,  the  water  forces 
its  way  through  the  small  hole  at  the  bottom  of  the  tube  and 
A  p          insinuates  itself  between  the  tube  and  the  sac,  with  the  result 

FIG.  141  —Phisalix'    *nat  *^e  latter  ig  easily  separated. 

guides  for  collodion  (£)  insertion  into  the  peritoneal  cavity.— Collodion  sacs 
may  be  used  in  experiments  upon  guinea-pigs,  rabbits, 
dogs,  sheep,  cattle  [and  birds  (fowls  and  pigeons)].  All  these  animals 
tolerate  aseptic  sacs  filled  with  sterile  broth  very  well.  The  technique  of 
the  operation  is  described  above  (p.  174,  B). 

After  an  interval  varying  from  a  few  days  to  several  months,  the  animal 
is  killed  and  the  sac  withdrawn  and  its  contents  investigated.  When  the 
sac  has  been  in  the  peritoneal  cavity  several  weeks  it  not  infrequently  happens 
that  it  is  found  to  be  broken ;  [even  then  it  is  in  the  case  of  birds  at  least 
usually  covered  with  a  fibrous  sheath  which  prevents  dispersal  of  the  con- 
tents]. It  is  well  therefore  to  use  several  animals,  to  be  sure  of  finding  at 
least  one  sac  intact.  To  remove  the  contents,  sterilize  the  bottom  with  a 
hot  wire,  insert  a  pipette  and  aspirate  the  fluid. 

D.  Reed  sacs. — Roux  and  Nocard  suggested  using,  in  place  of  collodion, 
a,  small  piece  of  the  tubular  membrane  lining  the  central  canal  of  the  bul- 
rush. Sacs  made  with  this  membrane  are  more  permeable  than  those  made 
of  collodion. 

1.  Take  a  few  pieces  of  common  bulrush  and,  if  they  are  fresh   boil   them  in 
water  for  about  a  quarter  of  an  hour,  but  if  dry  autoclave  them  at  115°  C.  for  an 
hour  instead. 

2.  After  softening  them  sharpen  one  end  in  the  same  way  as  a  lead  pencil,  in 
order  to  expose  the  membrane  lining  the  central  canal.     This  membrane  is  then 
carefully  denuded  for  a  certain  length. 

3.  Tie  one  end  of  the  separated  membrane  firmly  like  a  purse,  then  by  pressing 
gently  on  this  end  with  a  glass  rod  it  can  be  turned  inside  out. 

4.  Tie  a  small  glass  tube  into  the  open  end  and  fasten  it  with  a  stout  ligature, 
and  place  another  ligature  on  the  sac  itself  below  the  end  of  the  tube.     Fill  the 
sac  with  water  and  sterilize  as  in  the  case  of  collodion  sacs. 

5.  Suck  up  the  water  out  of  the  sac  and  replace  it  with  the  culture,  tie  the  ligature 
on  the  reed  and  disconnect  the  sac  from  the  tube  above  this  ligature.     Cover  each 
ligature  with  a  drop  of  melted  gum  lac. 

Introduce  into  the  peritoneal  cavity  in  the  manner  already  described. 


INOCULATION  INTO   THE   BILIARY  PASSAGES         177 

(ix)  Inoculation  into  the  biliary  passages. 

In  all  animals  in  common  use  for  experimental  purposes  the  bile  is  poured 
into  the  duodenum  through  a  simple  channel — the  bile  duct — of  which  the 
orifice  is  more  or  less  close  to  the  pylorus.  In  the  dog  the  opening  is 
4-12  cm.,  in  the  rabbit  about  1  cm.,  beyond  the  pylorus,  and  in  the 
guinea-pig  about  the  middle  of  the  duodenum.  The  operation  in  the  rabbit, 
guinea-pig  and  dog  will  be  described.  The  strictest  asepsis  must  be 
observed. 

A.  Guinea-pig.    Rabbit. — 1.  Anaesthetize  the  animal  and  fasten  it  on  its 
back.     Shave  the  hair  and  cleanse  the  skin  of  the  abdomen. 

2.  Make  an  incision  about  6  cm.  long  in  the  middle  line  commencing  about 
1  cm.  below  the  xiphoid  cartilage.     Cut  through  the  skin  and  aponeurosis, 
stop  any  bleeding,  then  incise  the  peritoneum  on  a  director. 

3.  Identify  the  pyloric  end  of  the  stomach,  then,  using  the  index  finger, 
find  the  duodenum  and  bring  it  to  the  surface.     The  opening  of  the  bile  duct 
will  be  seen  about  its  centre. 

4.  Having  identified  the  canal,  isolate  and  fix  it  on  a  blunt  hook.     Pass 
the  end  of  a  fine  needle  bent  at  a  right  angle  very  obliquely  through  the 
wall  and  in  the  line  of  its  long  axis.     Inject  the  material. 

5.  Withdraw  the  needle,  and  touch  the  point  where  it  penetrated  the  wall 
with  a  sponge  soaked  in  carbolic  water.     Stitch  up  the  aponeurosis  with  silk, 
suture  the  skin  and  paint  the  incision  with  collodion. 

B.  Dog. — 1.  Anaestnetize  the  animal  and  fix  it  on  its  back.     Shave  the 
hair  and  cleanse  the  skin  of  the  abdomen. 

2.  Make  a  longitudinal  incision  in  the  middle  line,  or  a  little  to  the  right, 
about  8  cm.  long,  commencing  a  few  centimetres  below  the  xiphoid  cartilage. 
Cut  through  the  skin  and  aponeurotic  layer,  and  stop  any  haemorrhage. 
Incise  the  peritoneum  on  a  director. 

3.  With  the  first  finger  in  the  wound,  follow  the  lower  surface  of  the  liver, 
then  bend  the  finger  to  hook  up  the  duodenum  and  bring  the  latter  to  the 
surface,  and  to  the  left. 

4.  Find  the  right  edge  of  the  duodenum,  and  follow  it  until  the  finger 
meets  the  fold  of  the  lesser  omentum  in  which  the  bile  duct  lies  with  the  portal 
vein,  hepatic  artery  and  nerves.     The  duct  lies  superficially  in  the  fold  and 
can  be  recognized  by  its  pearly  appearance,  its  structure,  its  direction,  and 
by  the  fact  of  its  opening  into  the  duodenum  at  a  distance  of  from  4  to  12  cm. 
beyond  the  pylorus. 

5.  Isolate  the  duct  on  a  small  director  and  fix  it  in  a  blunt  hook.     Pass 
the  bent  needle  very  obliquely  through  the  wall  in  the  line  of  its  long  axis. 
Inject  the  material,  and  complete  the  operation  as  above. 

(x)  Inoculation  into  the  portal  vein. 

The  operation  is  easier  in  the  dog,  but  can  also  be  done,  though  with  some 
difficulty,  in  the  guinea-pig  and  rabbit.  The  walls  of  the  vein  are  very  thin, 
and  easily  torn.  The  technique  described  above  for  finding  the  bile  duct 
is  applicable  to  the  isolation  of  the  portal  vein  in  the  dog.  It  is  better  to 
operate  as  follows  : 

1.  Anaesthetize  the  animal,  and  fix  it  on  its  left  side.     Shave  and  cleanse 
the  skin  of  the  part. 

2.  Make  an  oblique  incision  in  the  right  hypochondrium  commencing  above 
at  the  junction  of  the  last  rib  with  the  vertebral  column  and  extending  to 
the  outer  edge  of  the  rectus  muscle  at  the  crest  of  the  ileum.     Cut  through 

M 


178  EXPERIMENTAL  INOCULATIONS 

the  skin  and  muscular  layers.     Stop  the  haemorrhage.     Incise  the  peritoneum 
on  a  director. 

3.  Get  an  assistant  to  pass  his  fingers  into  the  wound  and  push  the  intestines 
to  the  left  as  far  as  possible  and  hold  them  in  the  abdomen. 

4.  Deep  down,  in  the  upper  part  of  the  wound  below  the  liver  the  bend  of 
the  duodenum  will  be  seen,  and,  on  a  level  with  it,  the  principal  mesenteric 
veins  converging  above  towards  the  portal  vein. 

5.  Having  recognized  the  vein,  isolate  it,  fix  it  and  perforate  the  wall  with 
a  bent  needle.     Inject  the  material. 

6.  Withdraw  the  needle  and  wipe  the  puncture  in  the  vein  with  a  sponge 
soaked  in  carbolic  water.     Put  in  two  layers  of  sutures  and  cover  the  skin 
incision  with  collodion. 

(xi)  Inoculation  into  the  kidneys. 

The  dog,  rabbit,  guinea-pig,  etc.,  may  be  used  for  this  experiment. 

1.  Lay  the  animal  on  the  side  opposite  to  that  on  which  it  is  proposed  to 
operate,  and  anaesthetize  it.     Shave  and  cleanse  the  skin  over  the  region 
to  be  operated  upon. 

2.  Make  an  incision  outside  the  sacro-lumbar  muscles  from  the  anterior 
end  of  the  last  rib  to  the  sacrum. 

3.  Incise  the  muscles  for  the  whole  length  of  the  skin  incision  on  a  level 
with  the  external  border  of  the  floor  of  the  lumbar  region. 

4.  Retract  the  margins  of  the  wound  widely  and  the  peri-renal  adipose 
tissue  will  be  exposed  behind  the  peritoneum.     Then,,  after  tearing  through 
the  loose  cellular  adipose  tissue  with  a  director,  the  kidney  appears  in  the 
wound. 

5.  Push  the  needle  into  the  renal  parenchyma  and  make  the  injection. 
Withdraw  the  needle  and  touch  the  needle  prick  with  a  sponge  soaked  in 
carbolic  water.     Insert  two  sets  of  sutures,  and  paint  the  skin  incision  with 
collodion. 

Ureter. — The  ureter  can  be  exposed  by  operating  in  the  same  way.  When  the 
kidney  is  freed  from  the  peri-renal  adipose  tissue  the  ureter  will  be  seen  lying  with 
the  renal  vessels  and  nerves. 

(xii)  Inoculation  into  the  anterior  chamber  of  the  eye. 

A.  Liquids. — 1.  Fix  the  animal  on  its  belly,  and  fasten  the  head  so  that 
the  animal  cannot  move  it.     It  is  well  to  anaesthetize  the  eye  by  dropping  into 
it  a  few  drops  of  a  1*5  per  cent,  solution  of  cocaine.     In  about  5  minutes 
the  eye  is  completely  insensitive. 

2.  Hold  the  eyelids  apart  and  fix  the  eye  with  the  thumb  and  index  finger 
of  the  left  hand.  Insert  the  needle  perpendicularly  to  the  axis  of  the  eye  at 
the  margin  of  the  cornea  at  the  corneo-sclerotic  junction.  Inject  a  few 
drops  of  the  fluid  and  withdraw  the  needle. 

B.  Solid.—!.  As  in  "  A  "  above. 

2.  Then,  the  eyelids  being  held  apart  and  the  eye  fixed  make*  an  incision 
a  few  millimetres  long  along  the  upper  border  of  the  cornea  with  a  cataract 
knife  or  very  fine  scalpel. 

3.  Holding  the  tissue  to  be  inoculated  with  a  fine  pair  of  bent  forceps 
pass  it  through  the  incision  and  force  it  as  far  as  possible  into  thelanterior 
chamber  by  rubbing  the  cornea  lightly  with  a  Daviel's  curette  or  the  blunt 
end  of  a  probe. 

Inoculations  into  the  anterior  chamber  are  generally  done  on  rabbits  and  are 
practised  chiefly  for  infecting  with  hydrophobia  or  syphilis,  or  for  studying  the 
development  of  tuberculosis  or  the  phenomena  of  phagocytosis. 


INOCULATION  INTO  THE  RESPIRATORY  PASSAGES     179 

(xiii)  Inoculation  into  the  respiratory  passages. 

A.  Inoculation  into  the  lungs. — 1.  Shave  and  cleanse  the  skin  of  the  thorax 
in  the  neighbourhood  of  the  axillary  fold. 

2.  Push  the  needle  perpendicularly  through  one  of  the  upper  intercostal 
spaces  to  a  depth  of  from  one  to  several  centimetres,  according  to  the  size 
of  the  animal.  Inoculate  the  material  and  withdraw  the  needle. 

B.  Intra-tracheal  inoculations  (mammalia). — 1.  Fix  the  animal  on  its  back 
with  the  head  extended  and  the  neck  raised  by  means  of  a  firm  plug  of  cotton- 
wool, a  large  india-rubber  cork  or  a  small  block,  etc.     Shave  and  cleanse  the 
skin  in  the  middle  line  below  the  larynx. 

2.  Make  an  incision  2-3  cm.  long  through  the  skin  in  the  middle  line  of  the 
neck  in  front  of  the  trachea. 

3.  Incise  the  aponeurosis  on  a  director. 

4.  Having  exposed  the  trachea,  pass  the  needle  obliquely  into  the  lumen 
between  two  of  the  cartilaginous  rings.     Inject  the  material.     Withdraw  the 
needle  and  wash  the  perforation  with  a  sponge  soaked  in  carbolic  water. 

Notes. — (a)  In  small  animals  it  is  convenient  as  soon  as  the  trachea  is  exposed 
to  fix  it  by  passing  a  suture  needle  threaded  with  silk  through  it. 

(6)  To  avoid  all  risk  of  inoculating  the  material  into  the  cellular  tissue  or  into  the 
walls  of  the  trachea  itself  the  following  precautions  may  be  taken.  Use  a  small, 
very  fine  trocar  with  a  cannula  which  should  be  shorter  than  the  syringe  needle. 
When  the  trachea  is  exposed  pass  the  cannula  between  two  of  the  cartilaginous 
rings,  withdraw  the  trocar  leaving  the  cannula  in  position.  Pass  the  syringe  needle 
through  the  cannula  so  that  the  point  of  the  needle  passes  beyond  the  end  of  the 
latter.  Make  the  injection  and  withdraw  the  needle  first,  then  the  cannula. 

5.  Suture  the  skin.     Cover  the  wound  with  collodion. 

C.  Intra-tracheal  inoculation  in  birds. — The   opening  of  the  trachea  is 
behind  the  base  of  the  tongue. 

1.  Open  the  beak  and  draw  the  tongue  forwards  with  a  pair  of  forceps. 

2.  The  opening  of  the  trachea  will  be  seen  behind  the  tongue  and  the 
material  to  be  inoculated  is  injected  straight  into  it. 

D.  Intra-pleural   inoculation. — Besson   has   shown   as   a   result   of  some 
observations  which  he  made  with  Pourrat  that  it  is  a  very  difficult  matter  to 
inoculate  a  fluid  into  the  pleural  cavity  without,  at  the  same  time,  injuring 
and  penetrating  the  lung.     Consequently  intra-pleural  inoculation   is   not 
very  exact. 

In  those  cases  where  the  syringe  needle  is  passed  obliquely  from  below 
upwards  through  an  intercostal  space  (6th  or  7th)  it  often  happens  either 
that  the  pleural  cavity  has  not  been  reached  or  that  the  needle  has  passed 
beyond  it.  To  perform  a  true  intra-pleural  inoculation  the  following 
technique  must  be  followed. 

1.  After  fixing  the  animal  on  its  left  side,  shave  and  cleanse  the  skin  over 
the  middle  of  the  right  side  of  the  thorax. 

2.  Make  an  incision  about  3  cm.  long  through  the  sixth  space  about  its 
centre  parallel  to  the  rib  and  passing  through  the  skin,  subcutaneous  cellular 
tissue  and,  if  desired,  the  external  intercostal  muscle. 

3.  Have  ready  a  blunt-pointed  needle,  laterally  perforated  and  previously 
sterilized  and  connected  to  a  syringe  by  means  of  an  india-rubber   tube 
(p.  167).     Pass  the  needle  into  the  intercostal  space,  directing  it  somewhat 
obliquely.     The  parietal  pleura  attached  to  the  outer  wall  of  the  cavity 
allows  the  needle  to  pass  through  :    the  visceral  pleura,  with  the  lung,  is 
driven  back  by  the  blunt  end  of  the  needle  and  when  the  latter  has  gone  a 


180 


EXPERIMENTAL  INOCULATIONS 


distance  of  from  a  few  millimetres  to  2  cm.  according  to  the  species  of  animal 
used,  the  operator  can  feel  that  it  is  moving  freely  in  a  cavity.  At  this  stage 
the  fluid  is  inoculated. 

4.  Withdraw  the  needle  quickly.     Stitch  up  the  skin  and  paint  the  wound 
with  collodion. 

E.  Inhalation. — 1.  Put  the  animal  in  a  solid- walled  metal  cage  having 
an  observation  window  on  one  side  and  on  the  other  two  holes  lightly  plugged 

with  cotton- wool  to  allow  of  interchange  of  air  : 
the  tube  from  the  pulverizer  passes  through  a 
third  hole. 

2.  Liquid  cultures  may  be  pulverized  by  means 
of  Richardson's  apparatus,  but  when  the  virus 
can  be  used  dry  without  losing  its  virulence  it 
is  better  to  pour  the  liquid  culture  on  to  lyco- 
perdon  spores,  lycopodium  powder,  or  on  to  wood 
charcoal  reduced  to  an  impalpable  powder.  The 
culture  is  intimately  mixed  with  the  powder  and 
then  dried  in  vacuo  over  concentrated  sulphuric 
acid.  The  powder  well  dried  is  then  pulverized 
in  the  cage  with  a  pair  of  bellows. 

Note. — When  dealing  with  micro-organisms  patho- 
genic for  man,  the  operator  should  be  particularly 
careful  to  protect  himself  from  the  dust,  and  the 
experiment  should  be  done,  for  choice,  in  the  open 
air. 

(xiv)  Intra-cranial  inoculation. 

Intra-cranial  inoculation  is  generally  performed 
on  the  dog,  guinea-pig  or  rabbit. 

A.— 1.  Fasten  the  animal  on  its  belly,  the  head 
being  firmly  held  by  an  assistant. 

2.  Shave"  and  cleanse  the  scalp  behind  the 
orbits. 

3.  Make   an  incision  through   the    skin  and 
aponeurosis  about  3  cm.  long  in  the  middle  line, 
commencing  at  a  point  level  with  the  upper 
borders  of  the  orbits.     Retract  the  edges  of  the 
incision  with  a  speculum.    In  the  dog  the  incision 
is  made  preferably  a  few  millimetres  from  the 
middle  line  to  avoid  the  superior  longitudinal 
sinus. 

4.  Place  a  small  trephine  about  5  millimetres 
in  diameter  (fig.  142)  on  the  skull  towards  the 
middle  of  the  incision  and  a  little  outside  the 
middle  line. 

Commence  trephining  and  when  the  teeth  bite 
raise  the  axis  to  prevent  wounding  the  brain. 

Ascertain  frequently  to  what  depth  the  trephine  has  reached  and  when 
resistance  has  ceased  raise  the  circle  of  bone  with  a  pair  of  forceps  or  small 
elevator. 

5.  The  dura  mater  is  now  visible  at  the  bottom  of  the  wound.     Pass  the 
needle  very  obliquely  through  the  meninges  so  that  the  brain  may  not  be 
injured,  and  inject  the  fluid.     It  is  well  to  use  a  needle  bent  to  a  right  angle 
at  the  middle  point  of  its  length. 


FIG.  142. — Trephine  for  small 
animals. 


INTRA-SPINAL  INOCULATION  181 

6.  Withdraw  the  needle,  touch  the  puncture  with  a  sponge  soaked  in 
carbolic  water,  suture  the  skin  and  apply  collodion  to  the  incision. 

B. — In  injecting  small  doses  of  toxin  into  the  cerebral  tissues  the  foregoing 
technique  may  be  simplified. 

After  shaving,  cleansing  and  incising  the  skin  make  a  small  hole  in  the 
skull  with  a  drill,  limiting  the  depth  of  the  perforation  with  a  shield  to 
avoid  damaging  the  meninges.  Then  the  needle  is  introduced  to  the 
required  depth  (determined  beforehand  by  means  of  a  probe)  the  toxin 
injected,  the  puncture  touched  with  carbolic  and  a  stitch  put  through  the 
skin  and  covered  with  collodion. 

(xv)  Intra-spinal  inoculation. 

Intra-spinal  inoculation  is  effected  through  the  posterior  occipito-atloid 
ligament.  With  a  little  practice  it  is  easy  to  inoculate  directly  into  the  spinal 
canal  of  a  rabbit  or  dog  by  forcing  a  curved  needle  into  the  ligament  through 
the  skin  (which  must  have  been  previously  cleansed) :  the  needle  passes 
behind  the  posterior  occipital  tuberosity  just  outside  the  middle  line  and 
is  then  turned  and  made  to  follow  the  occipital  bone.  Before  injecting 
make  sure  that  the  needle  has  passed  well  into  the  canal  by  aspirating  a 
little  of  the  cerebro-spinal  fluid  into  the  syringe  (which  should  be  not  quite 
full  of  the  material  to  be  injected).  The  inoculation  must  be  done  very 
slowly. 

It  is  easier,  especially  in  the  guinea-pig,  to  fix  the  animal  on  its  belly  with  the 
head  flexed,  j:hen  to  divide  the  posterior  cervical  muscles  transversely  on  a  very 
small  director  below  the  posterior  occipital  tuberosity,  avoiding  the  vertebral  veins. 
When  the  muscles  are  divided  the  ligament  will  be  seen  and  will  be  recognized 
by  its  pearly- white  appearance.  Bleeding  is  easily  arrested  by  plugging  the  wound 
with  wool  soaked  in  a  solution  of  peroxide  of  hydrogen.  By  keeping  close  to  the 
lower  surface  of  the  occipital  bone  the  membrane  is  easily  pierced  with  a  curved 
needle.  After  inoculating  insert  two  sets  of  sutures  and  paint  the  wound  with 
collodion. 

(xvi)  Inoculation  into  the  alimentary  canal. 

A.  Ingestion. — (a)  The  simplest  method  is  to  mix  the  culture  with  the 
animal's  food,  viz.  bran  in  the  case  of  rabbits  and  guinea-pigs  and  meal  in 
the  case  of  dogs. 

(b)  In  small  animals  the  culture  may  be  sucked  up  into  a  Pasteur  pipette 
which  is  then  introduced  into  the  animal's  mouth,  and  •  the  liquid  allowed  to 
fall  drop  by  drop  while  the  head  is  held  up.     The  end  of  the  pipette  should 
be  short  and  stout. 

[In  the  experiments  of  the  Royal  Commission  on  Tuberculosis  (1901)  it  was  found  that 
pipette-feeding  experiments  with  liquids  were  unsatisfactory.  The  animal  may  cough 
or  choke,  and  the  fluid  find  its  way  to  the  lungs.  Even  when  the  fluid  appeared  to  be 
swallowed  quite  satisfactorily  disease  of  the  lungs  was  sometimes  found  which  could 
not  have  been  of  intestinal  origin.  Experiments  conducted  on  these  lines  are  likely, 
therefore,  to  lead  to  erroneous  conclusions.] 

(c)  In    the    case    of   birds    make    small   pellets   of  flour   and    mix    the 
culture  with  it.     Put  a  pellet  on  the  base  of  the  tongue  and  close  the 
beak. 

B.  Oesophageal  catheterization. — This  method  is  more  certain  than  the 
foregoing  and  moreover  the  amount  of  liquid  injected  can  be  measured. 

Guinea-pigs  and  rabbits.— The  animal  with  the  head  moderately  extended 
is  held  by  an  assistant.  By  pressing  the  cheeks  near  the  molar  teeth  the 
mouth  can  be  opened  and  a  small  gag  with  a  hole  in  the  centre,  or,  better,  a 
piece  of  iron  wire  bent  into  a  rectangle,  can  be  placed  between  the  jaws  behind 


182  EXPERIMENTAL  INOCULATIONS 

the  incisor  teeth  (fig.  143).  A  very  fine  gum-elastic  catheter  can  then  be 
easily  passed  through  the  hole  in  the  gag  into  the  stomach.  By  attaching 
the  needle  of  the  syringe  to  the  open  end  of  the  catheter,  the  fluid  can  be 
injected. 

Dogs. — Fix  the  animal  on  its  back,  and  gag  it  as  described  at  p.  163.     Pass 
a  small  cesophageal  sound  or  a  rather  firm  piece  of  ordinary  india-rubber 
tubing  of   the  size  of  an  ordinary  pen-holder   into    the 
stomach.     Inoculate  the  culture  through  the    sound    or 
tubing. 

It  is  often  necessary  to  render  the  contents  of  the 
stomach  alkaline  before  introducing  the  culture  ;  this  can 
be  done  by  injecting  1  or  2  grams  of  sodium  bicarbonate 
dissolved  in  a  little  water. 

__  C.  Inoculation  into  the  intestines. — 1.  Open  the  ab- 

FIG.  i43.-Gag  for  ceso-   domen  as  described  on  p.  174. 

phageai  catheterization.        g.  Pick  up  and  hold  a  loop  of  intestine  with  a  pair  of 
forceps. 

3.  Pierce  the  wall  of  the  loop  obliquely  with  the  syringe  needle.     Inject 
the  material  and  withdraw  the  needle  at  once. 

4.  Dab  the  loop  with  a  sponge  soaked  in  carbolic  solution  :    suture  the 
aponeurosis  and  then  the  skin.     Paint  with  collodion. 

D.  Rectal  injection. — The  animal  must  be  firmly  held  by  an  assistant, 
then  with  a  stout  blunt-pointed  needle  inject  the  fluid  into  the  rectum. 


SECTION  VI.— OBSERVATIONS  TO  BE  MADE   ON  INOCULATED 

ANIMALS. 

In  studying  an  experimentally-induced  disease  the  symptoms  to  which  it 
gives  rise  in  the  inoculated  animal  should  be  observed  and  recorded  day 
by  day. 

A  note  should  be  made  of  the  following  points  : 

1.  The  local  lesion. — The  presence  or  absence  of  a  local  lesion.     The  time 
when  it  appears.      Its  situation,  extent,  nature  and  the  changes  which  it 
undergoes.     Enlargement  of  the  glands. 

2.  Temperature. — The  temperature  should  be  taken  at  least  twice  a  day 
in  the  rectum,  with  a  thermometer  graduated  in  tenths  of  a  degree  centigrade 
and  of  a  size  suitable  to  the  species  of  animal  under  observation.     The  tem- 
perature must  always  be  taken  before  inoculating  the  animal.     It  is  necessary 
to  bear  in  mind  that  all  animals  have  not  the  same  normal  temperature  ;  in 
guinea-pigs,  rabbits[,  goats,  pigs]  and  cattle 1  the  normal  temperature  varies 
from  38'5°  to  39'5°  C.,  that  of  horses  and  asses  between  38°  and  39°  C.  and 
of  birds  between  41°  and  42°  C.     In  small  animals,  complete  immobilization 
rapidly  reduces  the  temperature  which  should  therefore  never  be  taken  with 
the  animal  tied  to  the  operating  table.     A  curve  of  the  temperature  should 
be  kept. 

3.  Weight. — Animals   must   always    be    weighed    before    inoculation.     A 
ratio  can  then  be  established  between  the  weight  of  the  animal  and  the 
quantity  of  virus  which  must  be  inoculated  in  order  to  produce  sickness  or 
lead  to  death.     In  chronic  conditions,  the  animal  should  be  weighed  from  time 

[*  The  normal  temperature  of  an  adult  bovine  animal  is  usually  constant  in  the  neigh- 
bourhood of  38-5°  C. ;  that  of  a  young  calf  may  vary  fro-n  38'9°  C.  to  40'0°  C.  with  a 
mean  of  39'2°-39-40  C.] 


CLINICAL  OBSERVATIONS  183 

to  time  :  the  weight  curve  furnishes  valuable  information  as  to  the  course  of 
the  disease. 

4.  Auscultation. — The  development  of  pulmonary  lesions  can  be  detected 
and  followed  by  auscultation. 

5.  Condition  of  the  alimentary  canal. — Observation  should  be  kept  upon 
the  animal's  appetite  and  it  is  highly  important  to  notice  if  it  suffers  from 
diarrhoea,  etc. 

6.  Urine. — Does  the  urine  contain  pus,  blood  albumin,  etc.  ? 

7.  The  appearance  of  the  animal. — Whether  the  animal  is  lively  and  active 
or  dull  and  quiet,  the  condition  of  its  coat  whether  rough  and  badly  kept  or 
smooth  and  well  tended,  as  well  as  the  position  assumed  by  the  animal  (whether 
lying  on  its  side  or  curled  up)  are  all  important  facts  which  should  be  noted. 
The  appearance  of  twitchings,  convulsions  or  signs  of  paralysis  should  also 
be  carefully  watched  for. 

Observation  of  the  clinical  condition  must  be  subsequently  supplemented 
by  an  examination  of  the  tissues,  fluids  and  exudates  for  micro-organisms. 


CHAPTER   XL 
POST  MORTEM  EXAMINATIONS. 

Introduction. 

1.  Instruments,  p.  184.     2.  Preliminary    operations,    p.    185.     3.  Examination  of  the 

external  surface  of  the  carcase,  p.  185.     4.  Examination  of  the  internal  organs,  p.  185. 

5.  Removal  of  tissues  for  histological  examination,  p.  188. 

THE  objects  of  post  mortem  examination  are  two  in  number. 

1.  To  ascertain  as  far  as  possible  the  nature  of  the  lesions  which  were  the 
cause  of  death. 

2.  To  collect  material  for  further  investigation.     This  will  involve  the 
search  for  micro-organisms  in  the  blood,  exudates  and  internal  organs  by 
microscopical,  cultural  and  inoculation  methods,  as  well  as  the  histological 
examination  of  portions  of  the  internal  organs.     The  material  therefore  will 
have  to  be  collected  under  very  strict  aseptic  precautions. 

The  following  general  rules  must  be  observed  in  conducting  a  post  mortem 
examination  on  small  animals. 

A.  To  avoid  soiling  the  bench  fasten  the  animal  to  a  sheet  of  zinc  or 
copper  [or  pin  it  by  the  paws  to  a  sheet  of  cork  or  a  wooden  board  covered 
with  cork  linoleum,  either  of  which  can  be  washed  with  antiseptics,  preferably 
2  per  cent,  lysol,  before  and  after  use].     Lay  all  the  instruments  in  use  on 
the  metal  tray  [or  wooden  board]  and  not  on  the  bench  while  the  examination 
is  in  progress. 

B.  Use  sterile  forceps  and  not  the  fingers  for  raising  the  skin,  muscles 
and  internal  organs. 

C.  Sterile  instruments  must  be  used  throughout. 

D.  Conduct  the  examination  at  the  earliest  moment  possible  after  the 
death  of  the  animal. 

E.  As  soon  as  the  examination  is  completed  burn  the  carcase  and  any 
wool  or  paper  which  may  have  been  used  in  an  incinerator  (fig.  13,  p.  16) 
or  in  a  fire  with  a  good  draught,  boil  the  instruments,  and  if  a  metal  tray 
has  been  used  immerse  it  in  a  vessel  of  boiling  water  if  it  is  not  too  large, 
or  wash  it  with  a  strong  solution  of  lysol  or  carbolic  acid. 

1.  Instruments. 

Have  ready  before  commencing  a  post  mortem  examination — 

1.  Sterile  scalpels,  bistouries,   dissecting  forceps  and  scissors  both  large 
and  small. 

2.  A  number  of  sterile  Pasteur  pipettes. 


PRELIMINARY   OPERATIONS  185 

3.  Two  or  three  platinum  wires  one  of  which  should  be  stout  and  flattened 
at  the  end  in  the  form  of  a  small  spatula. 

4.  An  iron  rod  of  the  size  of  a  large  goose- quill  and  15-20  cm.  long, 
mounted  in  a  wooden  handle. 

5.  A  tray  of  zinc  or  copper  or  a  sheet  of  cork. 

6.  Sterile  absorbent  wool  in  a  glass  bottle  plugged  with  wool  and  some 
sterile  filter  paper. 

Cut  a  sheet  of  filter  paper  into  pieces  about  10  cm.  square,  wrap  them  in  a  piece 
of  ordinary  paper  and  sterilize  the  packet  in  the  autoclave. 

7.  An  enamelled  iron  bowl  or  glass  dish  containing  an  antiseptic  solution 
(O'Ol  per  cent,  corrosive  sublimate,  or  oxycyanide  of  mercury  [or  2  per  cent, 
lysol]). 

8.  A  Bunsen  burner  or  a  spirit  lamp. 

9.  Tubes  of  agar,  broth,  etc. 

10.  [Slides  and  cover-glasses.] 

11.  Wide-mouthed,  glass-stoppered  bottles  of  30-50  c.c.  capacity. 

2.  Preliminary  operations. 

1.  Fasten  the  animal  securely  to  the  tray.     In  the  case  of  rabbits,  guinea- 
pigs,  cats,  etc.,  lay  them  on  the  back,  pass  a  slip  knot  round  each  paw  and 
tie  to  holes  in  the  sides  of  the  tray  [or  if  a  wooden  board  be  used  pin  the 
animal's  extended  paws  to  it  with  large  drawing  pins]. 

Frogs,  mice,  sparrows,  etc.,  can  be  pinned  down  on  their  backs  to  the  cork 
sheet,  one  pin  being  passed  through  the  neck  the  others  through  the  extended 
paws  or  wings. 

In  the  case  of  fowls  and  pigeons  cut  off  the  wings,  lay  the  animal  on  its 
back  and  fasten  the  neck  and  legs  by  cords  passed  through  holes  in  the  sides 
of  the  tray. 

2.  The  animal  being  fastened  out,  thoroughly  wet  the  surface  of  the  thorax 
and  abdomen  with  the  antiseptic  and  cut  off  the  hair  gathering  up  the  loose 
hair  in  a  piece  of  paper  which  is  then  burnt.     Never  cut  off  the  hair  without 
first  of  all  wetting  it. 

In  the  case  of  birds  the  same  precaution  must  be  adopted  before  plucking 
the  feathers. 

3.  Examination  of  the  external  surface  of  the  carcase. 

Before  opening  the  carcase,  examine  the  external  surface  carefully  for 
lesions  of  the  skin,  abscesses,  etc.  If  an  abscess  be  found,  cut  away  the  hair, 
cauterize  the  surface  thoroughly  with  a  red-hot  iron  rod,  flame  and  break 
off  the  point  of  a  Pasteur  pipette  as  quickly  as  possible,  push  the  pipette 
through  the  centre  of  the  cauterized  area  and  aspirate  the  pus  through  the 
plugged  end. 

If  the  pus  be  thick  and  inspissated — as  it  often  is  in  the  case  of  rabbits — and 
cannot  be  drawn  into  the  pipette,  make  an  incision  with  a  sterile  knife  after  cauterizing 
the  skin  and  collect  the  contents  either  on  the  point  of  the  knife  or  with  a  stout 
platinum  wire. 

After  sowing  two  or  three  tubes  of  culture  media  with  some  of  the  material 
prepare  films  for  microscopical  examination. 

4.  Examination  of  the  internal  organs. 

As  a  rule  it  is  better  to  open  the  thorax  first.  If  the  abdomen  were  opened 
before  the  thorax  it  might  happen  that  contamination  of  the  thoracic  organs 
could  not  be  prevented. 


186 


POST   MORTEM  EXAMINATIONS 


A.  Mammalia. 

1.  Pick  up  the  skin  over  the  manubrium  sterni,  incise  it  and  prolong  the 
incision  to  the  lower  part  of  the  abdomen,  then  extend  the  incision  outwards 
to  the  roots  of  the  four  limbs.  Dissect  the  skin  from  the  subjacent  tissues 
and  throw  the  flaps  outwards.  This  incision  must  involve  the  skin  only. 
'  2.  Should  there  be  reason  to  suspect  the  presence  of  excess  of  fluid  in  the 
pleural  cavities,  cauterize  the  muscles  over  one  of  the  intercostal  spaces, 

C.L.G. 


-A.L.G. 


, \r-I.L.G. 


FIG.  144. — Appearance  presented  by  a  normal  guinea-pig. 

C.L.G.  Cervical  lymphatic  glands;  A.L.G.  Axillary  lymphatic  glands; 
M.G.  Mesenteric  gland ;  I.L.G.  Inguinal  lymphatic  glands ;  P.M.L.G.  Post- 
manubrial  lymphatic  gland  ;  P.S.L.G.  Post-sternal  lymphatic  glands. 

introduce  the  point  of  a  flamed  pipette,  aspirate  some  of  the  fluid,  sow  cultures 
and  make  films. 

3.  To  open  the  thorax.     Kaise  the  tip  of  the  xiphoid  cartilage  with  a  pair 
of  sterile  forceps,  introduce  the  point  of  a  pair  of  strong  scissors  beneath  the 
costal  cartilages  a  little  to  one  side  of  the  sternum,  and  inclining  the  scissors 
slightly  outwards  cut  through  the  costal  cartilages  as  far  as  the  clavicle  and 
then  divide  the  clavicle.     By  doing  the  same  on  the  other  side  of  the  sternum 
a  flap  is  formed  which  can  either  be  turned  upwards  or  detached.     This  will 
expose  the  heart  and  lungs. 

4.  If  there  be  any  fluid  in  the  pericardium,  take  hold  of  the  latter  with  a 
pair  of  sterile  forceps,  flame  the  point  of  a  pipette  and  push  it  through  the 
membrane  close  to  the  forceps.     The  hot  end  of  the  pipette  will  sterilize  the 
surface  of  the  pericardium  as  it  passes  through  it.     Aspirate  the  fluid. 

5.  To  collect  blood  from  the  heart,  tear  through  the  pericardium  with 
two  pairs  of  forceps,  or  holding  it  with  a  pair  of  forceps  slit  it  up  with  a  pair 


EXAMINATION  OF  THE  INTERNAL  ORGANS  187 

of  fine  scissors.  Cauterize  the  surface  of  the  ventricle  with  a  red-hot  rod, 
pass  a  pipette  through  the  sterilized  area  and  aspirate  the  blood. 

6.  To  collect  material  from  an  hepatized  or  congested  area  of  the  lung, 
cauterize  the  surface  of  the  latter  and  pass  a  pipette  or  the  bent  end  of  a 
stout  platinum  wire  into  the  affected  part :   or  the  latter  may  be  exposed  by 
taking  hold  of  the  lung  with  two  pairs  of  sterile  forceps  and  tearing  it. 

7.  When  the  examination  of  the  thorax  is  completed,  open  the  abdomen. 
To  collect  the  peritoneal  fluid  lift  up  the  muscular  wall  with  a  pair  of 

forceps,  make  a  small  slit  with  a  sterile  scalpel,  introduce  a  pipette  through 
the  opening  and  aspirate  the  fluid  from  the  flanks.  The  pipette  should 
be  held  parallel  to  the  abdominal  wall  so  as  to  avoid  damaging  the 
intestine. 

Complete  the  incision  of  the  abdominal  wall  along  the'  middle  line  and 
throw  the  flaps  outwards. 

8.  Note  carefully  the  appearances  presented  by  the  internal  organs.     In 
taking  material  from  the  liver,  spleen,  kidneys  or  lymphatic  glands,  first 
cauterize  the  surface,  then  pass  a  stout  wire  bent  in  the  form  of  a  hook 
through  the  centre  of  the  cauterized  area  deeply  into  the  organ,  twist  it  round 
and  round  and  on  withdrawing  it  sow  the  material  at  once  on  a  suitable 
culture  medium.     For  making  films,  simply  tear  off  a  small  piece  of  the  organ 
with  a  pair  of  forceps  (Chap.  XIII.). 

To  examine  the  intestinal  contents,  cauterize  the  surface,  pass  a  pipette 
through  the  cauterized  area  and  aspirate  some  of  the  contents.  Urine  may 
be  collected  from  the  bladder  in  a  similar  manner,  a  ligature  being  first  tied 
round  the  urethra. 

9.  Bone  marrow. — To  examine  the  bone  marrow  expose  one  of  the  long 
bones,  divide  it  across  with  a  pair  of  strong  sterile  scissors  and  collect  the 
medulla  in  a  pipette  or  platinum  loop. 

If  the  bone  be  divided  with  non-sterilized  scissors  the  cut  end  must  be 
cauterized  with  a  heated  iron  rod  before  aspirating  the  medulla. 

10.  Examination   of  the  central  nervous  system. — Lay  the  body  on  its 
ventral  surface  and  fasten  the  feet  firmly  to  the  tray  [or  board]  as  before. 

Make  an  incision  through  the  skin  from  the  root  of  the  nose  to  the  sacrum 
along  the  line  of  the  spinous  processes  of  the  vertebrae,  and  reflect  the  skin  ; 
detach  the  scapulae  at  their  humeral  articulations  and  turn  them  on  one 
side  ;  then  dissect  away  the  masses  of  muscle  from  the  vertebral  laminae 
with  a  strong  bistoury,  taking  care  in  so  doing  not  to  penetrate  the  abdominal 
cavity  in  the  lumbar  region.  With  a  pair  of  curved  Liston's  forceps  (fig.  145) 


FIG.  145. — Curved  Liston's  forceps. 

open  the  skull  along  an  horizontal  line  passing  through  the  superciliary  ridges ; 
free  these  ridges  on  each  side  by  an  oblique  incision  ;  then  raise  the  frontal 
bone  with  an  elevator  and  detach  it  with  the  Liston's  forceps.  This  will 
expose  the  brain.  Having  reached  the  occipital  foramen  raise  the  spinous 
processes  with  the  elevator  and  cut  through  the  laminae  of  the  bodies  of  the 
vertebrae  with  the  forceps  alternately  on  the  right  and  left  sides.  This  if 
properly  done  (a  certain  amount  of  skill  and  patience  is  required  to  avoid 


188  POST  MORTEM  EXAMINATIONS 

injury  to  the  spinal  cord)  will  remove  the  spinous  processes  in  the  form  of  a 
rosary  held  together  by  the  ligamenta  flava. 

If  there  is  any  meningeal  exudate,  cauterize  the  surface  of  the  membrane, 
introduce  a  pipette  through  the  centre  of  the  cauterized  area  and  aspirate 
the  fluid. 

To  remove  portions  of  the  nerve  tissue  tear  through  the  meninges  with 
two  pairs  of  forceps,  cauterize  the  area  (cerebrum,  cerebellum,  medulla 
oblongata  or  spinal  cord),  and  push  the  point  of  a  strong  pipette  deeply  into 
the  tissue  ;  then  aspirate  the  material,  twisting  the  pipette  about  if  necessary. 
Or,  after  cauterization,  portions  of  tissue  may  be  removed  with  a  platinum 
loop  or  with  a  sterile  bistoury. 

B.  Birds. 

To  open  the  thorax  in  birds  it  is  best  to  divide  the  skin  along  the  middle 
line,  and  after  reflecting  it  to  each  side  to  make  a  curved  incision,  extending 
to  the  bone,  round  the  sternum ;  beginning  at  the  root  of  the  neck,  con- 
tinue along  the  right  margin,  round  its  lower  end  and  up  the  left  margin. 
Cut  through  the  clavicle  on  each  side  with  a  pair  of  stout  scissors,  and 
following  the  line  of  the  incision  through  the  soft  parts  detach  the  sternum 
from  the  ribs,  then  cut  away  the  muscular  attachments  and  remove  the 
breast  plate. 

The  examination  will  then  be  proceeded  with  as  in  the  former  case. 

C.  Post-mortem  examination  of  human  bodies. 

The  technique  to  be  employed  in  the  collection  of  material  post  mortem 
from  the  human  subject  does  not  differ  from  that  already  described  in  the 
case  of  animals,  and  the  methods  of  examination  should  also  be  the  same, 
but  it  must  be  remembered  that  if  the  results  of  the  bacteriological 
investigation  are  to  be  relied  upon  the  examination  must  be  made  within 
a  few  hours  of  death  ;  if  it  be  delayed  until  the  interval  required  by  law 
has  elapsed  (24  hours  after  death)  the  bacteriological  findings  must  be 
accepted  with  caution  especially  in  summer;  the  presence  of -the  colon 
bacillus  in  particular  in  the  internal  organs  would  be  under  such  circum- 
stances without  significance,  since  this  organism  multiplies  in  the  tissues  of 
the  body  immediately  after  death  and  sometimes  even  during  the  period 
immediately  preceding  death. 

Note. — The  material  collected  post  mortem  may  either  be  examined  and  sown  at 
once,  or  may  be  put  aside  for  examination  at  a  later  stage,  provided  that  both  ends 
of  the  pipettes  containing  the  material  be  sealed.  In  the  latter  case  to  reach  the 
contents  of  a  pipette,  push  down  the  wool  plug  almost  as  far  as  the  top  of  the  fluid, 
and  cut  off  the  part  of  the  pipette  above  it  with  a  glass-cutter  and  a  point  of  red 
hot  glass  ;  the  plug  can  then  be  taken  out  and  the  contents  manipulated  with  a 
pipette  just  as  in  the  case  of  a  culture- tube. 

5.  Removal  of  tissues  for  histological  examination. 

For  purposes  of  subsequent  histological  examination,  small  pieces  of  the 
internal  organs  [and  other  tissues]  should  be  removed  at  the  time  of  the  post 
mortem  examination.  The  pieces  should  be  quite  small  (cubes  of  10-15  mm.), 
but  should  be  cut  off  with  a  sharp  sterile  bistoury  so  that  the  section  may 
be  as  clean  as  possible.  Place  the  pieces  at  once  in  ground-glass  stoppered 
bottles  containing  one  of  the  following  fixing  solutions : 

1.  Absolute  alcohol. — For  bacteriological  purposes  absolute  alcohol  is  the 
simplest  and  the  most  generally  useful  fixative. 

The  method  of  placing  the  tissue  in  the  first  instance  in  weak  alcohol  and 


HISTOLOGICAL  EXAMINATION  189 

subsequently  transferring  to  solutions  of  increasing  strength  not  only  takes 
more  time  but  yields  only  moderate  results. 

To  fix  in  alcohol,  place  the  tissue  (about  1  cm.  cube)  in  25-30  c.c.  of  absolute 
alcohol :  renew  the  alcohol  after  3  hours  and  again  after  24  hours.  The 
tissue  is  then  fixed,  but  it  is  found  to  stain  better  if  left  in  the  alcohol  for 
3  days.  Tissues  should  not  under  any  circumstances  remain  in  absolute 
alcohol  for  more  than  a  week  at  the  outside,  and  if  it  is  not  convenient  to 
use  them  then  they  should  be  transferred  to  90  per  cent,  alcohol. 

The  tissue  should  be  suspended  in  the  fluid  or  laid  on  a  piece  of  wool  at  the 
bottom  of  the  bottle  to  ensure  its  hardening  uniformly. 

2.  Formalin. — Formalin  is  an  excellent  hardening  agent ;    it  does  not 
interfere  with  any  of  the  staining  methods  and  is  particularly  valuable  for 
tissues  which  are  to  be  cut  by  the  freezing  process.     The  best  solution  is 
the  following  : 

Commercial  formalin,       -  10  c.c. 

Distilled  water,        -  90     „ 

The  tissue  is  fixed  in  about  6-8  hours  ;  but  better  preparations  are  obtained 
if  the  formalin  is  allowed  to  act  for  24-48  hours. 

If  frozen  sections  are  to  be  cut  the  tissue  is  used  straight  out  of  the 
formalin  :  but  before  embedding  in  paraffin,  transfer  the  tissue  first  to  90  per 
cent,  alcohol,  then  to  absolute  alcohol,  leaving  it  for  24  hours  in  each  solution. 

3.  Corrosive  sublimate. — Corrosive  sublimate  is  another  most  useful  harden- 
ing agent,  and  can  be  used  whatever  method  of  staining  is  to  be  subsequently 
employed.     It  can  be  used  as  a  cold  saturated  solution1  but  it  penetrates 
and  fixes  the  tissue  better  when  acidified  with  acetic  acid. 

Acid  sublimate  (Mayer}. 

Saturated  aqueous  solution  of  corrosive  sublimate,      -         -       100  parts. 
Glacial  acetic  acid,  -         -         -       1-3      „ 

Allow  20-30  c.c.  for  each  piece  of  tissue.  The  solution  penetrates  well  and 
rapidly,  so  that  the  pieces  may  be  relatively  large  (cubes  of  2  cm.).  Leave 
in  the  acid  solution  for  at  the  most  12  hours.  The  tissue  is  then  white  and 
opaque.  Wash  in  running  water  for  an  hour  (this  is  not  absolutely  essential). 
Transfer  to  100  c.c.  of  70  per  cent,  alcohol  containing  xv-xx  drops  of  tinc- 
ture of  iodine  and  leave  for  24  hours  to  remove  the  excess  of  perchloride  and 
prevent  the  deposition  of  crystals  in  the  tissue.  Transfer  to  80  per  cent, 
alcohol  for  24  hours  and  finally  to  90  per  cent,  alcohol  for  a  similar  period. 

It  must  be  remembered  of  course  that  perchloride  of  mercury  acts  on  metal 
instruments,  so  that  in  removing  tissues  from  perchloride  solutions  horn,  glass  or 
wood  spatulas  must  be  used. 

4.  Flemming's  solution. — For  purposes  of  bacteriological  examination  the 
diluted  solution  is  better  than  the  concentrated. 

(a)  Diluted  solution. 

1  per  cent,  aqueous  solution  of  chromic  acid,  25  volumes. 

1  per  cent.         „  r,         osmic  acid,  -     ,     -         10          „ 

1  per  cent.         ,,  „         acetic  acid,  -         10          „ 

Distilled  water,  -  55 

(b)  Strong  solution. 

1  per  cent,  aqueous  solution  of  chromic  acid,      -  -         15  volumes. 

2  per  cent.         ,,  „        osmic  acid,  4          „ 
Glacial  acetic  acid,  1  volume. 

1  Cold  water  dissolves  about  6 '6  per  cent,  of  perchloride  of  mercury.  A  saturated 
solution  is  easily  prepared  by  dissolving  70  to  75  grams  of  perchloride  in  1  litre  of  distilled 
water  in  the  warm :  filter  while  warm,  and,  as  the  solution  cools,  white  needles  crys- 
tallize out  at  the  bottom  of  the  vessel ;  pour  off  the  supernatant  liquid  which  is  then 
ready  for  use. 


190  POST   MORTEM   EXAMINATIONS 

These  solutions  ought  to  be  prepared  just  before  use.  The  use  of  Flemming's 
solution  should  be  limited  to  the  hardening  of  nerve  tissues,  and  the  pieces 
should  be  very  small.  Many  stains  cannot  be  used  after  Flemming's  solu- 
tion ;  the  best  to  use  are  haematoxylin,  safranin  and  the  basic  aniline  dyes. 

Suspend  the  tissue  in  the  solution  and  leave  it  for  36-72  hours  (weak  solu- 
tion) or  1-24  hours  (strong  solution),  wash  in  running  water  for  24  hours, 
transfer  to  distilled  water  for  1  hour,  and  then  for  24  hours  to  each  of  the 
following  solutions  successively,  viz.  70  per  cent.,  80  per  cent.,  90  per  cent, 
alcohol. 

5.  Flemming-perchloride  solution. — A  mixture  of  acid  perchloride  and 
Flemming's  solution  combines  the  advantages  of  both.  The  mixture  is 
prepared  according  to  the  following  formula  : 

Saturated  aqueous  solution  of  perchloride  of  mercury,  -  500  c.c. 

1  per  cent,  aqueous  solution  of  chromic  acid,      -  -  500     „ 

Osmic  acid  crystals  1  gram. 

Glacial  acetic  acid,  -  50  c.c 

Harden  for  12—24  hours.  Wash  and  transfer  to  alcohol  as  in  the  case  of 
Flemming's  solution. 


CHAPTER   XII. 

THE  COLLECTION  OF  MATERIAL  FOR  BACTERIO- 
LOGICAL EXAMINATION. 

1.  Hair,  p.  191.  2.  Skin,  p.  191.  3.  Sputum,  p.  191.  4.  Blood,  p.  192;  collection  of 
serum,  p.  196.  5.  Pharyngeal  exudates,  p.  197.  6.  Abscesses,  p.  197.  7.  Aqueous 
humour,  p.  197.  8.  Pleural  and  pulmonary  exudates,  p.  198.  9.  Ascitic  fluid, 
p.  198.  10.  Tumours  and  lymphatic  glands,  p.  198.  11.  Splenic  puncture  and 
splenectomy,  p.  198.  12.  Lumbar  puncture,  p.  199.  13.  Milk,  p.  201.  14.  Urine, 
p.  201.  15.  Stools,  p.  202. 

1.  Hair. 

Man  and  animals. 

Pull  out  a  few  hairs  with  a  pair  of  sterile  forceps,  lay  them  between  two 
sterile  microscope  slides  and  wrap  up  the  slides  in  a  piece  of  sterile  paper. 
They  can  thus  be  put  aside  or  be  transmitted  to  the  laboratory  without  fear 
of  contamination. 

2.  Skin. 

Man  and  animals. 

1.  Cut  off  the  hair  with  a  pair  of  sharp  scissors. 

2.  Scrub  with  a  nail  brush  and  soap,  wash  with  boiled  water  and  Tub 
briskly  with  a  sponge  wrung  out  in  a  1  in  1000  solution  of  sublimate,  wash 
with  absolute  alcohol,  then  with  ether  and  wipe  quickly  with  a  piece  of 
sterile  filter  paper. 

3.  Pick  up  a  small  fold  of  the  skin  with  a  pair  of  sterile  forceps  and  cut  it 
off  through  the  base  with  a  sharp-pointed  sterile  bistoury. 

If  the  skin  be  thick  or  adherent  to  the  deeper  tissues  it  will  be  difficult  to 
pick  up  a  piece  of  the  size  required.  In  that  case  mark  out  a  small  rectangular 
area  with  the  bistoury,  detach  one  comer  and  then,  taking  hold  of  the 
latter  with  a  pair  of  sterile  forceps,  dissect  the  piece  of  skin  from  the  deeper 
tissues. 

4.  If  the  material  be  collected  at  the  bed-side  it  can  be  taken  to  the  labora- 
tory between  two  sterile  watch-glasses  or  in  a  sterile  glass  dish  wrapped 
up  in  paper. 

3.   Sputum. 

Man. 

A.  Ordinary  method  of  collection. — For  the  ordinary  microscopical  examina- 
tion of  sputum  for  the  tubercle  bacillus,  it  will  suffice  if  the  patient  cough  the 
sputum  into  a  sterile  bottle  or  clean  pocket  handkerchief.  The  material 
should  be  examined  as  soon  as  possible. 


192 


THE  COLLECTION   OF  MATERIAL 


B.  Kitasato  s  method. — This  method  is  much  to  be  preferred  when  cultures 
are  to  be  sown  or  investigations  of  a  more  delicate  nature  are  to  be  made. 

1.  The  patient  rinses  his  mouth  and  gargles  the  back  of  his  throat  several 
times  with  boiled  water  and  then  coughs  the  sputum  into  a  sterile  Petri  dish. 

2.  Transfer  the  sputum  immediately  to  a  tube  containing  several  cubic 
centimetres  of  sterile  water  and  shake  it  up  well.     Remove  the  sputum  from 
the  tube  with  a  sterile  platinum  loop  or  a  pair  of  sterile  forceps  to  a  second 
tube  of  sterile  water  and  wash  it  in  this  way  three  or  four  times  to  free  it, 
as  far  as  possible,  from  contaminating  organisms  (but  note  that  sputum  can 

only  be  washed  when  it  is  tenacious  and  lumpy  as  in  influenza, 
advanced  tuberculosis  (nummular  sputum),  etc.). 

3.  After  washing  spread  the  sputum  in  a  thin  layer  in  a  sterile 
Petri  dish  and  cut  off  a  small  fragment  with  a  small  pair  of 
sterile  scissors  or  platinum  needle  from  as  near  the  centre  as 
possible.  Use  this  for  sowing  cultures. 

4.  Blood. 

Man. 

A.  Pricking  the  skin. — A  small  quantity  of  blood  is  readily 
obtained  by  pricking  the  distal  end  of  the  finger  near  the  nail 
and  collecting  the  drops  in  some  suitable  sterile  vessel  such  as 
a  Pasteur  pipette,  a  small  tube  [or  a  Wright's  capsule  ]  or  on  a 
glass  slide.  This  method  is  however  only  applicable  when  the 
blood  is  required  for  immediate  microscopical  examination,  e.g. 
for  anthrax  bacilli,  hsematozoa,  etc.,  as  it  is  liable  to  con- 
tamination during  collection.  When  the  blood  is  required  for  sowing 
cultures,  it  should  be  taken  from  a  vein. 


FIG.  146  — 

Wright's  cap- 
sule for  collect- 
ing blood. 


FIG.  147. — Method  of  collecting  blood  by  pricking  the  finger. 

1.  Sc-rub    the   ball   of   the   finger    with   soap    and   wrater.      Wash    it    in 
perchloride,  alcohol  and  ether.     Dry  with  sterile  paper. 

2.  Compress  the  base  of  the  finger  by  grasping  it  with  the  left  hand  or  by 
tying  a  ligature  round  it  (fig.  147). 


BLOOD  193 

3.  With  a  sterile  pin  or   small  lancet  [or  a  straight  surgical  triangular 
needle]  prick  the  skin  sharply  and  deeply. 

4.  Wipe  away  the  first  drop  or  two  of  blood  which  issues  with  a  piece  of 
sterile  paper  and  collect  the  remainder. 

As  a  further  precaution  the  skin  of  the  finger  after  being  washed  and  dried 
may  be  painted  over  with  a  very  thin  layer  of  collodion.  The  finger  is  then 
pricked  through  the  collodion  and  in  this  way  the  blood  is  prevented  from  coming 
in  contact  with  the  skin. 

B.  Cupping. — A  larger  volume  of  blood  can  be  obtained  by  cupping,  but 
otherwise  the  method  is  open  to  the  same  objections  as  the  foregoing. 

1.  Asepticize  about  10  sq.  cm.  of  the  skin  of  the  thorax,  back  or  sides. 

2.  Apply  a  sterile  cupping-glass  over  the  part. 

3.  When  the  glass  has  fixed  itself,  raise  it  (the  operator's  hands  having,  of 
course,  been  already  sterilized),  scarify  the  skin  with  a  sterile  razor  and  apply 
the  cupping-glass  again. 

4.  When  sufficient  blood  has  collected  put  the  patient  in  such  a  position 
that  the  blood  will  not  be  spilt,  then  lift  the  glass  and  cover  it  at  once  with 
sterile  paper. 

C.  Bleeding  from  a  vein  at  the  bend  of  the  elbow. — By  this  method  all 
danger  of  contaminating  the  blood  is  avoided,  and  it  should  be  adopted  in 
all  cases  when  cultures  have  to  be  sown.     It  is  attended  by  no  danger  and  is 
less  painful  than  the  foregoing  methods. 

1.  Procure  a  sterilizable  syringe  of  2-20  c.c.  capacity  according  to  the 
amount  of  blood  to  be  collected,  and  adjust  a  sharp  and  clean-bored  needle. 
Test  its  efficiency  and,  if  satisfactory,  sterilize  it  with  the  needle  attached,  by 
boiling  it  in  water  for  15  minutes  or  by  heating  in  the  autoclave  at  115°  C. 

2.  Lay  the  patient's  forearm  flat  on  the  bed,  and  get  an  assistant  to  com- 
press the  middle  of  the  arm  or  put  a  tight  bandage  round  it  as  in  the  operation 
for  bleeding. 

3.  Wash  the  skin  over  the  bend  of  the  elbow  with  soap  and  rinse  with 
sublimate  or  oxycyanide,   then  with  alcohol  and    finally  with   ether.     As 
the  result  of  the  combined  compression  and  friction  the  veins  at  the  bend 
of  the  elbow  will  stand  out  prominently. 

To  make  certain  of  asepsis  it  is  sometimes  advised  to  lightly  touch  the  point 
through  which  the  needle  is  to  be  passed  with  a  cautery ;  but  in  the  great  majority 
of  cases  it  is  sufficient  to  wash  the  arm  in  the  manner  described. 

4.  Select  the  largest  vein,  push  the  needle  through  the  skin  and  then  into  the 
vein.    The  vein  lying,  as  it  does,  immediately  beneath  the  skin  is  generally  pene- 
trated at  the  same  time  as  the  skin.     The  needle  should  be  held  parallel  to  the 
long  axis  of  the  vein  and  at  a  very  acute  angle  to  the  surface.     When  the  vein 
is  entered,  by  gently  withdrawing  the  plunger,  blood  will  flow  into  the  syringe. 

Notes. — There  is  nothing  to  be  gained  by  pointing  the  needle  towards  the  hand ; 
on  the  contrary,  it  is  easier  to  point  it  towards  the  arm  and  the  calibre  of  the  vein 
is  such  that  it  flows  just  as  easily  into  the  needle  whichever  way  the  latter  is  directed. 
The  alternative  method  which  consists  in  first  making  an  incision  through  the  skin 
and  exposing  the  vein  should  never  be  practised. 

5.  When  the  syringe  is  full,  withdraw  the  needle  from  the  vein,  relieve 
the  pressure  and  apply  a  drop  of  collodion  to  the  needle  prick.     Be  careful 
not  to  let  the  blood  clot  in  the  syringe  but  squirt  it  at  once  into  a  sterile 
test-tube  ;  then  wash  the  syringe  in  cold  water  and  sterilize  it. 

Horses,  asses  and  cattle. 

In  these  animals  the  jugular  vein  is  the  most  convenient  from  which 
to  bleed.  The  method  has  already  been  described  in  dealing  with  the 

N 


194  THE   COLLECTION   OF  MATERIAL 

preparation   of  serum  (p.   48).     When  a  small  quantity  only  of  blood  is 
wanted  a  syringe  is  used  instead  of  a  trocar. 

Guinea-pigs. 

Guinea-pigs  may  be  bled  from  the  jugular  vein,  from  the  femoral  or  carotid 
arteries,  or  by  cardiac  puncture. 

A.  From  the  jugular  vein. — For  the  anatomical  data  see  p.  172. 

1.  Fix  the  guinea-pig  on  its  back  with  the  head  extended.     Shave  the 
skin  over  the  front  of  the  neck  and  cleanse  it  in  the  ordinary  way. 

2.  Make  an  incision  through  the  skin  and  sub -cutaneous  tissue  along  the 
line  of  the  vein,  dissect  away  the  cellular  tissue  with  a  director  and  the  vein 
will  come  into  view. 

3.  Pass  the  needle  of  a  sterile  syringe  or  the  end  of  a  pipette  (similar  to  that 
described  at  p.  166)  very  obliquely  into  the  vein.     If  a  slip  knot  be  passed 
under  the  vein  with  a  Deschamps  needle  on  the  cardiac  side  of  the  puncture, 
the  vessel  can  be  compressed  and  the  flow  of  blood  into  the  pipette  facilitated. 

4.  Having  collected  the  blood,  withdraw  the  needle  or  pipette  and  make 
certain  that  there  is  no  haemorrhage  from  the  puncture.     If  the  vein  be 
bleeding,  tie  a  ligature  above  and  below  the  puncture.     Put  two  or  three 
stitches  in  the  skin  and  cover  the  wound  with  collodion. 

Note. — The  blood  may  be  collected  directly  in  a  sterile  tube  or  flask  by  passing 
a  fine  trocar  into  the  exposed  vein.  The  operation  in  this  case  is  described  at 
p.  49. 

B.  Carotid  and  femoral  arteries. — 1.  Expose  the  vessel  (pp.  173  and  174). 

2.  Puncture  the  wall  of  the  artery  obliquely  with  a  syringe  needle,  the 
end  of  a  bent  pipette  or  a  small  trocar. 

3.  Having  collected  the  blood,  withdraw  the  instrument,  stitch  up  the  skin 
and  paint  the  incision  with  collodion. 

Sometimes  haemorrhage  occurs  when  the  needle  is  taken  out  of  the  artery.  This 
can  be  guarded  against  by  placing  two  ligatures  beneath  the  vessel,  one  above  and 
the  other  below  the  puncture,  then,  if  haemorrhage  occur,  the  two  threads  can  be 
tied  and  the  wounded  part  of  the  vessel  isolated. 

C.  Cardiac    puncture. — Cardiac    puncture    as    practised    in    physiological 
laboratories  may  be  usefully  applied  for  bacteriological  purposes  (Pagniez). 
It  allows  a  much  larger  volume  of  blood  to  be  collected  than  is  possible  by 
other  methods,  is  easily  performed  and  is  unattended  by  danger  to  the  animal ; 
moreover  the  blood  is  not  exposed  to  any  risk  of  contamination.     The  tech- 
nique, which  is  as  follows,  has  been  worked  out  by  Raybaud  and  Hawthorn. 

1.  Tie  down  the  animal  on  its  back,  shave  and  cleanse  the  skin  over  the 
front  of  the  cardiac  area.     Have  ready  a  sterile  syringe  capable  of  holding 
5  c.c.  and  fitted  with  a  needle  of  the  ordinary  pattern  but  very  sharp. 

2.  At  a  point  on  the  left  margin  of  the  sternum,  about  8-10  mm.  above 
the  angle  formed  by  the  base  of  the  xiphoid  cartilage  and  the  last  rib  carti- 
lage articulating  with  the  sternum,  push  the  needle  sharply  to  a  depth  of 
15-17  mm.  above  the  last  but  one  or  last  but  two  chondro -sternal  articulations. 

The  needle  will  pass  into  the  left  ventricle,  and  by  inclining  it  a  little  towards 
the  middle  line  it  can  be  made  to  enter  the  right  ventricle.  This  method  is  to  be 
recommended  because  the  risk  of  wounding  the  anterior  margin  of  the  left  lung  is 
lessened,  and  if  the  heart  were  punctured  at  a  higher  level  than  that  described  the 
auricle  would  be  penetrated  and  ruptured. 

3.  Fill  the  syringe  slowly  with  blood,  and  withdraw  the  needle  sharply 
and  quickly. 

Rabbits. 
A.  The  ear  veins. — The  simplest  method  of  collecting  blood  from  a  rabbit 


BLOOD  195 

is  to  take  it  from  one  of  the  veins  of  the  ear.     An  adult  rabbit  can  easily  be 
bled  to  the  extent  of  20  c.c.  in  this  way. 

1.  Prepare  a  large  Pasteur  pipette  with  the  pointed  end  short  but  strong 
and  bent  at  an  obtuse  angle  to  the  shaft  (fig.  133,  p.  166).     The  point  must  be 
sharp   and  have   thin  cutting  edges.     Sterilize  the  pipette  by  passing  it 
through  the  flame  but  be  careful  to  allow  it  to  cool  before  using  it.     In  this 
particular  case  a  pipette  is  better  than  a  syringe. 

2.  Let  the  animal  sit  on  the  knees  of  the  operator  or  of  an  assistant.     Shave 
the  hair  over  the  line  of  the  marginal  vein  and  cleanse  the  skin  in  the  ordinary 
way  (p.  172).     Compress  the  vein  at  the  root  of  the  ear  between  the  finger 
and  thumb  or  with  a  pair  of  pressure  forceps. 

3. "Holding  the  ear  in  the  left  hand,  thrust  the  point  of  the  pipette  through 
the  skin  and  then  into  the  lumen  of  the  vein.  .  A  flow  of  blood  into  the 
pipette  will  indicate  when  the  point  is  in  the  vein.  The  point  of  the  pipette 
should  be  directed  towards  the  tip  of  the  ear  and  must  be  held  absolutely 
parallel  to  the  axis  of  the  vein  to  avoid  penetrating  both  walls. 

The  flow  of  blood  into  the  pipette  is  slow  :  sometimes  it  ceases,  owing  to 
the  formation  of  a  small  clot  in  the  end  of  the  pipette  ;  this,  however,  can 
easily  be  displaced  by  aspirating  at  the  plugged  end  of  the  pipette. 

It  is  a  good  practice  to  puncture  the  vein  near  the  root  of  the  ear  so  that  if  unsuc- 
cessful at  the  first  trial  another  attempt  may  be  made  nearer  the  tip.  By  bleeding 
from  the  two  ears  in  turn,  blood  may  be  collected  at  frequent  intervals  from  the 
same  animal. 

4.  When  sufficient  blood  is  collected  remove  the  pipette  and  seal  the  point  in 
the  flame.     The  blood  can  afterwards  be  aspirated  into  other  Pasteur  pipettes 
through  the  plugged  end,  the  plug  being  well  flamed  before  being  taken  out. 

5.  Clip  the  wound  with  a  pair  of  pressure  forceps  for  a  moment  to  stop 
any  haemorrhage.     After  being  bled  the  animal  will  be  thirsty,  and  some 
water  should  be  left  in  its  cage. 

B.  Jugular  vein.— The  anatomical  data  and  the  technique  of  the  operation 
are  the  same  as  in  the  case  of  the  guinea-pig  (p.  194). 

C.  Carotid  and  femoral  arteries. — Here  again  the  description  given  for  the 
guinea-pig  is  applicable  (p.  194). 

D.  Cardiac   puncture. — The   technique   is   similar   to   that   described   for 
cardiac  puncture  in  the  guinea-pig.     C.  Nicolle  and  Duclaux  recommend 
using  a  rather  large  needle,  about  2  cm.  long,  fitted  to  a  sterile  syringe  of 
10-20  c.c.  capacity. 

1.  The  animal  is  held  down  on  its  back  and  the  skin  over  the  heart  shaved 
and  cleansed. 

2.  The  needle  with  syringe  attached  is  driven  in  sharply  to  a  depth  of 
17-18  mm.  to  the  left  of  the  sternum  in  the  fourth  intercostal  space  counting 
upwards  from  the  xiphoid  cartilage.     The  needle  must  be  inclined  from  below 
upwards  and  slightly  inwards. 

3.  Aspirate  the  blood  slowly  into  the  syringe  and  then  withdraw  the 
needle  quickly. 


Dogs  are  most  easily  bled  from  the  jugular  or  external  saphenous  vein 
(p.  173),  or  from  the  carotid  or  femoral  artery,  the  ordinary  rules  of  asepsis 
being  observed.  It  is  to  be  remembered  that  dogs'  blood  coagulates  very 
quickly. 

Birds. 

Bleed  from  the  axillary  vein  (p.  173)  adopting  the  ordinary  aseptic 
precautions. 


196 


THE  COLLECTION  OF  MATERIAL 


Collection  of  serum. 

On  account  of  the  importance  at  present  attaching  to  a  study  of 
serum  reactions  it  is  often  necessary  to  collect  serum  from  immunized 
animals.1  In  the  case  of  large  animals  it  is  best  to  bleed  by  the  method 
described  on  p.  49.  Small  animals  may  be  bled  preferably  from  the  carotid 
by  the  method  just  described,  and  after  the  clot  has  contracted  the  serum 
can  be  decanted.  But  by  this  method  much  of  the  serum 
is  lost,  being  retained  in  the  meshes  of  the  clot  and  it  is 
better,  therefore,  when  the  amount  of  blood  available  is 
strictly  limited  as  is  the  case  with  small  animals,  to  bleed 
into  a  Latapie's  tube.  By  using  this  apparatus  all  chance 
of  contaminating  the  blood  is  avoided  and  a  yield  of  80  per 
cent,  of  the-  total  volume  of  serum  is  assured. 

Latapie's  apparatus.  —  This  (fig.  148)  consists  of  a  large  glass 
tube  B  constricted  about  its  lower  third  E,  and  having  a  small 
cup  F  at  its  lower  end.  Below  the  constriction  there  are  two 
tubulures,  one  T,  straight  and  open  and  plugged  with  wool  between 
two  constrictions  ;  the  other  D,  on  the  opposite  side,  bent  in  the 
form  of  an  inverted  U,  and  drawn  out  at  its  free  end  to  a  fine 
point  which  is  sealed.  The  upper  end  of  the  large  tube  B  is 
connected  by  means  of  a  piece  of  india-rubber  tubing  with  another 
tube  A,  known  as  the  trocar  tube,  consisting  of  an  ordinary  test- 
tube  bent  in  the  form  of  a  right  angle  and  drawn  out  to  a  fine  point 
at  its  free  end.  This  second  tube  A  passes  well  down  into  the 
larger  tube  B,  leaving  the  bent  and  pointed  end  projecting. 
Occupying  the  centre  of  the  apparatus  from  top  to  bottom  is  a 
small  glass  tube  H,  sealed  at  one  end  and  the  sides  perforated  with 
numerous  holes. 

Technique.  —  Place  a  few  drops  of  water  in  the  apparatus  and 
sterilize  it  in  the  autoclave  at  120°  C.     Ex- 
pose the  carotid  in   the  ordinary   way  then 

break  off  the  point  of  the  tube  A  with  a  pair  of  sterile  forceps 

and  pass  it  into  the  vessel,  holding  the  apparatus  so  that  the 

broken  point  is  downwards.     Blood  will  now  ascend  into  the 

tube  A.     Stop  the  flow  of  blood  before  the  latter  is  quite  full, 

then  seal  the  pointed  end  of  A  in  the  flame,  gently  aspirating 

through  T  to  prevent  the  blood  being  clotted  by  the  heat. 

Stand  the  apparatus  on  one  side  with  the  tube  A  downwards. 

The  clot  forms  around  the  narrow  central  tube  H,  and  re- 

tracts from  the  walls  of  A.     If  the  apparatus  be  now  inverted 

the  serum  will  fall  into  the  collecting  bulb  R,  the  red  cells 

precipitating  into  the  cup  F.     In  this  way  80  per  cent,  of  the 

serum  can  be  collected  in  a  few  hours,  and  can  be  easily  with- 

drawn  through   the   tubulure   D  by  breaking  its  point  and 

blowing  through  T.     With  a  little  experience  and  skill  a  small 

animal  such  as  a  rabbit  or  guinea-pig  can  be  bled  two  or  three 

times  without  killing  it. 

Stassano's  apparatus.  —  Stassano's   apparatus    is    somewhat 

similar  to  Latapie's  but  is  fragile  and  more  expensive. 

Lumiere's  tube.  —  This  consists  of  a  glass  tube  (fig.  149)  on 

which  two  bulbs  B  and  D  are  blown,  the  interior  of  the  lower 

B  having  a  number  of  projecting  points.     The  tube  is  plugged 

with  wool  at  the  ends  and  sterilized  in  the  hot  air  sterilizer. 

To  use  the  apparatus  the  tube  A  is  fitted  with  a  short  piece 

of  india-rubber  tubing  carrying  a  sterilized  platinum-iridium 

syringe  needle.     As  soon  as  the  vessel  is  penetrated,  blood 

will  flow  into  the  bulb  B.     When  the  latter  is  full,  the  tubing 

is  pinched  and  the  needle  withdrawn  from  the  vessel.     The 

1  The  collection  of  serum  for  use  as  a  culture  medium  is  described  in  Chapter  II. 


FIG.  148.  — La- 
tapie's apparatus 
for  collecting  blood 
from  small  animals . 


TONSILLAR   EXUDATES  197 

tube  A  is  then  tilted,  the  india-rubber  tubing  detached,  and  after  passing  it 
through  the  flame  the  end  A  is  plugged  with  wool.  When  the  blood  is  clotted 
the  apparatus  is  inverted,  the  clot  will  be  held  by  the  points  in  B  and  the  serum 
will  run  into  the  bulb  D. 

Centrifuging. — The  maximum  yield  of  serum  is  obtained  in  the  minimum  of 
time  by  centrifuging  the  blood  (Camus).  Collect  the  blood,  without  contaminating 
it,  in  a  number  of  sterile  centrifuge  tubes  (vide  infra),  plug  the  tubes  with  wool 
and  centrifuge  at  once.  The  serum  collects  in  the  upper  part  of  the  tube  and  the 
clot  below.  If  the  animal  was  fasting  at  the  time  of  bleeding,  the  serum  will  be 
clear  and  transparent ;  on  the  other  hand  if  digestion  was  going  on  the  serum  will 
be  milky  and  slightly  opaque. 

5.  Pharyngeal  exudates. 
Man. 

A.  Puncture  of  the  tonsil. — 1.  Get  the  patient  to  clean  the  surface  of  the 
mucous  membrane  by  thoroughly  rinsing  out  his  mouth  with  boiled  water. 

2.  Make  the  patient  sit  up  and  incline  his  head  at  a  suitable  angle,  then 
press  the  tongue  down. 

3.  Take  a  rather  long  stout-pointed  Pasteur  pipette  with  a  sharp  cutting 
end,  heat  it  well  in  the  flame  and  then  pass  it  rapidly  and  deeply  into  the 
tissue  of  the  tonsil.     The  heated  end  cauterizes  and  sterilizes  the  surface  of 
the  gland  and  is  itself  cooled  before  reaching  the  deeper  parts.     Aspirate 
lightly  through  the   plugged   end  of  the  pipette  and  then  withdraw  the 
instrument. 

4.  The  small  quantity  of  material  which  will  be  obtained  should  be  sown 
at  once  into  broth  and  the  pipette  washed  out  two  or  three  times  by  aspirating 
some  of  the  broth  and  blowing  it  out  again. 

B.  False  membranes. — After  the  patient  has  washed  out  his  mouth  with 
boiled  water,  press  the  tongue  down  and  strip  off  the  false  membrane  with  a 
pair  of  sterile  forceps.     If  the  membrane  be  friable  it  may  be  that  the  forceps 
will  not  pick  it  up,  in  which  case  it  can  be  removed  by  rubbing  it  with  a 
plug  of  sterile  wool  held  in  a  pair  of  forceps  or  affixed  to  an  iron  wire. 

When  the  membrane  is  detached  it  should  be  blotted  firmly  between 
two  pieces  of  sterile  filter  paper  to  remove  any  contaminating  organisms, 
that  may  be  on  the  surface. 

6.  Abscesses. 

Man. 

1.  Cleanse  and  if  necessary  shave  the  skin. 

2.  Puncture  the  abscess  with  a  needle  of  large  calibre  and  aspirate  the  pus 
into  a  sterile  syringe. 

3.  If  the  pus  be  inspissated  and  cannot  be  aspirated  in  this  way,  make  a 
small  incision  through  the  skin,  introduce  the  end  of  a  large  Pasteur  pipette 
and  aspirate  the  pus  into  the  pipette,  or  collect  some  of  it  with  a  platinum 
loop. 

Animals. 

1.  Shave  the  hair  and  cauterize  a  small  area  of  the  skin  over  the  abscess. 

2.  Pass  a  Pasteur  pipette  through  the  centre  of  the  eschar  and  aspirate 
the  pus. 

7.  Aqueous  humour. 
Animals. 

1.  Fix  the  animal  so  that  it  cannot  move  and  keep  the  eyelids  retracted 
with  a  speculum.  Wash  the  conjunctiva  with  warm  sterile  water. 


198  THE  COLLECTION   OF  MATERIAL 

2.  Hold  the  eye  firmly  between  the  thumb  and  index  finger  of  the  left  hand, 
and  with  a  screwing  movement  pass  a  fine  Pasteur  pipette  perpendicularly 
to  the  axis  of  the  eye  at  the  margin  of  the  cornea  into  the  anterior  chamber. 
The  fluid  will  ascend  into  the  pipette  without  aspiration. 

8.  Pleural  and  pulmonary  exudates. 

Man  and  animals. 

A  small  quantity  of  a  pleural  effusion  can  easily  be  collected  with  a  sterile 
syringe.  Use  a  long  needle  (5-7  cm.)  with  a  large  bore.  When  the  exudate 
consists  of  thick  and  granular  pus  it  is  better  to  use  a  small  trocar  attached 
to  a  suitable  syringe. 

1.  Asepticize  the  skin  ;   to  make  quite  sure  of  the  asepsis  the  site  through 
which  the  needle  is  to  pass  may  be  superficially  cauterized. 

2.  Pass  the  needle  mounted  on  a  syringe  into  one  of  the  intercostal  spaces 
and  aspirate  the  fluid  into  the  syringe. 

3.  Transfer  the  material  to  a  sterile  test-tube. 

These  small  punctures  are  quite  unattended  by  danger  and  may,  if  necessary, 
be  repeated. 

The  same  technique  may  be  employed,  when  there  is  no  fluid  in  the  pleura, 
for  puncturing  the  lung  to  reach  (for  example)  a  pneumonic  patch  previously 
delimited  by  auscultation.  In  this  case  a  fine  needle  is  passed  perpendicularly 
and  more  or  less  deeply  through  one  of  the  intercostal  spaces  and  a  little 
blood-stained  fluid  aspirated  into  the  syringe. 

9.  Ascitic  fluid. 

Man. 

A  large  volume  of  ascitic  fluid  may  •  be  collected  aseptically  by  using  a 
trocar  with  sterile  rubber  attachment.  The  fluid  is  best  collected  in  a  sterile 
bottle  covered  with  paper.  The  operation  must  be  carried  out  under  the 
ordinary  surgical  conditions  and  the  rules  for  puncture  of  the  abdomen 
observed. 

The  fine  trocar  of  a  Potain's  apparatus  with  the  india-rubber  adjustments 
on  its  lateral  tubulure  is  very  useful  for  the  purpose. 

10.  Tumours  and  lymphatic  glands. 

Tumours  and  lymphatic  glands  must  be  removed  in  the  ordinary  surgical 
manner,  strict  asepsis  being  maintained  and  care  being  taken  that  the  struc- 
ture is  not  touched  with  the  hands. 

When  the  tumour  or  gland,  as  the  case  may  be,  is  enucleated,  sterilize .  a 
small  area  of  the  surface  with  a  well-heated  iron  wire,  pass  a  sterile  platinum 
needle  or  bistoury  through  the  cauterized  part  and  remove  the  material 
required  from  the  centre. 

11.  Spleen. 
Splenic  puncture  in  man. 

The  spleen  has  been  punctured  for  the  purpose  of  recovering  the  typhoid 
bacillus  from  patients  suffering  from  enteric  fever  and  is  sometimes  practised 
in  the  study  of  certain  other  infections,  e.g.  Leishmanioses,  etc. 

1.  Delimit  the  spleen  by  percussion  and  asepticize  the  skin. 

2.  Use  a  long  needle  (5  cm.)  attached  to  a  syringe  by  a  piece  of  india- 
rubber  tubing  (p.  167)  and  penetrate  the  skin  perpendicularly  over  the  centre 
of  the  spleen.     Aspirate,  withdraw  the  needle,  and  paint  the  puncture  with 
collodion. 

3.  A  few  drops  of  blood  generally  represent  the  material  collected.     This 


LUMBAR  PUNCTURE  199 

is  sown  into  broth  by  drawing  some  sterile  broth  into  the  syringe  and  expelling 
it  again  into  the  culture-tube. 

The  india-rubber  connexion  is  absolutely  necessary  :  it  allows  the  needle 
to  follow  the  movement  of  the  spleen  and  so  avoids  any  risk  of  tearing  the 
organ. 

Splenic  puncture  is  not  often  performed  and  is  not  altogether  unaccom- 
panied by  danger. 

Splenectomy  in  animals. 

The  functions  of  the  spleen  in  the  resistance  of  the  body  to  certain  infectious 
diseases  can  be  studied  by  observation  of  the  results  following  the  removal 
of  the  organ.  Dogs  and  rats  are  the  best  animals  for  the  experiment,  but 
the  operation  can  be  performed  on  many  other  species. 

The  spleen  is  situated  in  the  left  flank  beneath  the  lower  false  ribs  and 
near  the  left  curvature  of  the  stomach. 

1.  Fix  the  animal  on  its  right  flank  and  anaesthetize  it. 

2.  Shave  and  asepticize  the  skin  of  the  left  flank.     Sterilize  all  instruments 
and  asepticize  the  hands  carefully. 

3.  Make  an  incision  a  few  centimetres  long  through  the  skin  and  sub- 
cutaneous tissues  immediately  below  the  margin  of  the  last  rib,  commencing 
at  the  angle  and  continuing  parallel  to  the  bone. 

4.  Cut  through  the  aponeurosis  of  the  external  oblique  and  then  of  the 
internal  oblique  on  a  director. 

5.  Separate  the  fibres  of  the  transversalis  with  the  blunt  end  of  a  director. 

6.  Incise  the  peritoneum  for  the  whole  length  of  the  incision. 

7.  The  spleen  will  then  be  exposed  or  can  readily  be  found  by  passing  the 
finger  along  the  greater  curvature  of  the  stomach  :   draw  it  out  of  the  wound, 
being  very  careful  not  to  lacerate  it. 

8.  Tear  through  the  gastro-splenic  omentum  and  put  a  firm  silk  ligature 
around  the  vessel  of  the  hilum.     Cut  through  the  pedicle  on  the  distal  side 
of  the  ligature. 

9.  Suture  the  muscles,  close  the  skin  wound  with  a  few  stitches  and  cover 
the  incision  with  collodion. 

12.  Lumbar  puncture. 

Man. 

By  means  of  lumbar  puncture,  an  operation  devised  by  Essex  Wynter, 
a  needle  can  be  passed  into  the  cerebro-spinal  canal  and  the  fluid  withdrawn. 
Bacteriological  investigation  of  the  cerebro- 
spinal  fluid  is  of  great  interest  and  importance 
in  cases  of  meningitis. 

Anatomical  data.— In  the  adult  the  spinal 
cord  only  reaches  to  the  lower  border  of  the  first 
or  upper  border  of  the  second  lumbar  vertebra, 
but  in  children  twelve  months  old  it  reaches  to 
the  level  of  the  third.  The  spinal  cord  cannot, 
then,  be  injured  by  passing  a  fine  trocar  into 
the  spinal  canal  through  the  third,  fourth  or 
fifth  lumbar  spaces.  In  these  situations  the 
nerves  comprising  the  cauda  equina  are  sus- 
pended in  the  cerebro-spinal  fluid  and  are 
collected  into  two  lateral  fasciculi  separated  by 

an  interval    of   5    mm.     The  lower  down   the         FlG.  150.-Landmark3  for 
puncture    is    made    the    smaller    the    chance    the  operation  of  lumbar  puncture 


200  THE   COLLECTION   OF   MATERIAL 

of  wounding  the  nerves  since  they  diminish  in  number  as  the  canal  is 
descended. 

The  transverse  width  of  the  third  and  fourth  lumbar  spaces  is  from 
18-20  mm.  and  their  depth  from  above  downwards  10-15  mm.  Their  shape 
varies  with  age  :  the  fifth  space  between  the  last  lumbar  arch  and  the  upper 
border  of  the  sacrum  is  wider  than  but  not  quite  so  deep  as  the  two  above 
and  marks  the  situation  of  the  inferior  arachnoidal  cul-de-sac,  which  is  a 
true  reservoir  of  cerebro-spinal  fluid. 

The  operation  is  generally  performed  between  the  fourth  and  fifth  lumbar 
vertebrae.  An  horizontal  line  drawn  between  the  highest  points  of  the  two 
iliac  crests  passes  through  the  tip  of  the  spinous  process  of  the  fourth  lumbar 
vertebra  ;  by  inserting  the  needle  immediately  below  this  process  the  space 
between  the  fourth  and  fifth  lumbar  vertebrae  is  entered. 

The  depth  to  which  the  needle  must  be  inserted  will  depend  upon  the 
age  and  also  upon  the  state  of  nutrition  of  the  patient ;  in  a  child  it  will  be 
1'5  cm.,  2  cm.  and  sometimes  3  cm.  according  to  its  condition ;  in  the  adult, 
4-6  cm.  If  the  needle  pass  too  far  it  may  reach  the  premeningeal  venous 
plexus  and  cause  a  slight  haemorrhage,  in  which  case  the  needle  must  be 
withdrawn  a  little  before  the  cerebro-spinal  fluid  can  be  collected. 

Operation. — 1.  Sterilize  a  platinum-indium  needle  with  a  short  bevel  and 
a  calibre  of  0*8-1  mm.  and  about  5  cm.  long  for  a  child  and  8  cm.  for  an 
adult. 

The  needle  should  have  a  fine  platinum  wire  passed  through  it  reaching  as 
far  as  the  bevel  but  not  interfering  with  its  cutting  edge. 

2.  Place  the  patient  in  the  lateral  decubitus  on  the  edge  of  the  bed  with 
the  thighs  strongly  flexed  on  the  abdomen  and  the  legs  on  the  thighs,  the 
head  being  slightly  raised  on  a  pillow  and  flexed  on  the  thorax. 

The  patient  may  also  sit  up  with  his  legs  hanging  over  the  edge  of  the 
bed,  the  body  being  bent  forward  and  the  back  arched.  This  position,  how- 
ever, though  more  convenient  for  lumbar  puncture,  is  often  rendered 
impossible  by  illness  and  has  the  disadvantages  of  tiring  the  patient  and 
stimulating  muscular  reaction. 

3.  Asepticize  the   skin  by  washing   with   soap,   ether  and  alcohol.     Or. 
more  simply,  paint  the  surface  of  the  skin  a  few  minutes  before  doing  the 
operation,  with  tincture  of  iodine.     The  surgeon   must  of  course  prepare 
his  hands  as  for  any  other  surgical  operation. 

4.  Determine  the  position  of  the  line  connecting  the  highest  points  of  the 
crests  of  the  iliac  bones  (vide  ante).     This  line  will  pass  through  the  upper 
border  of  the  spinous  process  of  the  fourth  lumbar  vertebra. 

5.  Put  the  tip  of  the  left  index  finger  on  the  spine  of  the  fourth  lumbar 
vertebra  and  keep  it  in  that  position  throughout  the  operation.     Take  the 
needle  with  the  platinum  wire  in  it  in  the  right  hand  and  pass  it  perpen- 
dicularly to  the  surface  immediately  below  the  spinous  process  and  very  near 
(not  more  than  1  cm.  away  from)  the  median  line,  slowly  but  deliberately 
into  the  spinal  canal.     Direct  the  needle  forwards  and  a  little  upwards. 
The  needle  will  pass  through  in  order,  the  lumbo-sacral  muscles,  the  ligamen- 
tum  flavum,  the  dura  mater  and  the  arachnoid  membrane.     As  soon  as  it 
enters  the  sub-arachnoid  space  the  liquid  will  issue  from  the  needle. 

6.  Withdraw  the  platinum  wire  and  collect  the  fluid  in  a  sterile  test- tube. 

7.  Collect  5  or  6  c.c.  in  the  case  of  a  child  and  10-15  c.c.  in  the  case  of  an 
adult.     Lumbar  puncture  is  unattended  with  danger  if  no  more  fluid  than 
this  be  aspirated.     Withdraw  the  needle  and  paint  over  the  puncture  with 
iodoform  and  collodion.     The  patient  should  remain  in  bed  for  24  hours 
after  the  operation. 


URINE  201 


Notes. — 1.  No  advantage  is  obtained  from  local  anaesthesia,  but  in  the  case  of 
very  nervous  patients  the  skin  may  be  sprayed  with  ethyl  chloride. 

2.  As  the  needle  passes  through  the  skin  there  is  "occasionally  a  reflex  muscular 
contraction  of  the  lumbo-sacral  muscles.     Should  this  occur  desist  for  a  few  moments 
before  continuing  to  push  the  needle  into  the  canal. 

3.  Should  the  needle  be  driven  against  bone  its  point  will  be  bent  and  another 
attempt  will  have  to  be  made  taking  a  better  direction. 

4.  If,  during  the  operation,  the  needle  becomes  obstructed  it  is  easily  cleared 
with  the  platinum  wire. 

5.  Occasionally  the  fluid  is  blood-stained  :    in  that  case  the  needle  has  wounded 
some  of  the  small  meningeal  veins  ;    this  is  a  matter  of  no  importance  and  can  be 
remedied  by  slightly  altering  the  position  of  the  needle. 

13.  Milk. 

Duclaux  adopts  the  following  technique  which,  though  delicate,  gives  a 
sterile  milk  without  any  heat : 

1.  Take  a  number  of  plugged  sterile  test-tubes. 

2.  Wash  and  brush  the  cow's  udder  with  soap  and  water,  rinse  with  per- 
chloride  of  mercury  then  with  alcohol  and  finally  with  sterile  water.     The 
milker  then  sterilizes  his  hands. 

3.  Reject  the  first  few  drops  of  milk,  which  serve  to  wash  the  walls  of  the 
excretory  canals. 

4.  An  assistant  takes  the  plug  out  of  one  of  the  tubes  and  holds  the  latter 
as  close  to  the  mouth  of  the  teat  as  possible  without  touching  it ;   when  the 
tube  is  half- full  he  replaces  the  plug.     A  number  of  tubes  may  be  filled  in 
the  same  way. 

14.  Urine. 

Man. 

To  collect  urine  in  a  sterile  manner  proceed  as  follows. 

1.  Take  a  red  rubber  catheter,  protect  .the  upper  end  with  a  small  cap  of 
filter  paper,  then  wrap  up  the  whole  instrument  carefully  in  several  folds 
of  paper  and  autoclave  for  20  minutes  at  115°  C.     On  taking  it  out  of  the 
autoclave  dry  it  in  the  incubator. 

2.  Put  the  man  on  his  back  and  carefully  wash  the  glans  and  meatus  with 
a  1  in  a  1000  solution  of  oxy cyanide  of  mercury,  sponge  with  wool  which  has 
been  sterilized  in  the  autoclave  and  wrap  the  penis  in  another  wool  sponge 
similarly  sterilized.     The  operator  now  sterilizes  his  hands. 

3.  Remove  the  catheter  from  its  paper  covering  by  taking  hold  of  its 
upper  end  ;   dip  the  other  end  in  oil  sterilized  at  115°  C. 

4.  Lay  the  catheter  for  a  moment  on  the  paper  in  which  it  was  sterilized. 
Hold  the  penis  in  the  left  hand,  and  pick  up  the  catheter  about  its  middle 
with  the  right,  introduce  it  into  the  meatus  and  push  it  along  the  urethra 
still  resting  the  upper  end  on  the  paper  which  should  be  held  by  an  assistant. 

5.  On  reaching  the  entrance  to  the  bladder  pinch  the  catheter  firmly  between 
the  thumb,  and  index  finger  and  pass  the  catheter  through  the  sphincter. 

6.  The  assistant  flames  the  mouth  of  a  flask  previously  sterilized  in  the 
hot  air  sterilizer,  removes  the  wool  plug  and  holds  the  mouth  to  the  end  of 
the  catheter  from  which  he  now  removes  the  paper  cap. 

7.  Relax  the  pressure  on  the  catheter  and  the  urine  will  flow  into  the 
flask.     When  the  latter  is  three-parts  filled  pinch  the  catheter  to  stop  the 
flow  of  urine.     The  assistant  flames  the  neck  of  the  flask  and  replaces  the 
wool  plug  which  he  has  been  holding  in  his  left  hand  during  the  time  the 
flask  has  been  filling. 

A  similar  technique  can  be  adopted  in  the  case  of  large  animals. 


202  THE   COLLECTION   OF  MATERIAL 

Small  animals  (rabbits,  guinea-pigs,  etc.). 

It  is  impossible  to  use  a  catheter  on  these  small  animals  and  the  only  way 
to  collect  the  urine  in  the  male  is  to  let  it  flow  into  a  sterile  tube  or  Pasteur 
pipette.  The  animal  should  be  fixed  on  its  back  and  the  emission  of  urine  is 
easily  provoked  by  laying  towels  wrung  out  in  very  cold  water  on  the  abdomen 
and  loins. 

15.  Stools. 

Stools  for  bacteriological  examination  should  be  collected  in  a  sterile 
vessel  and  care  must  be  taken  that  they  are  not  mixed  with  urine. 

When  solid,  cauterize  the  surface  with  a  red-hot  iron  rod  and  collect  some 
of  the  material  from  the  centre.  When  liquid,  take  up  the  quantity  required 
with  a  Pasteur  pipette  or  platinum  loop. 


CHAPTER  XIII. 

THE   BACTERIOLOGICAL  EXAMINATION   OF 
FLUIDS  AND  TISSUES. 

Section  I. — Film  preparations,  p.  203. 

1.  Unstained  preparations,  p.  203.     2.  Stained  preparations  :  A.  Preparation  of 
films — (a)  Fluids,  p.  204;    (6)  Scrapings  of  organs,  p.   205;    (c)   Sputum,   p.   205. 
B.  Staining  methods  :    (a)  Simple  staining,  p.  205  ;   (6)  Differential  staining,  p.  207. 
Section  II. — Histological  preparations,  p.  211. 

1.  Instruments,  p.  211.  2.  Freezing  methods,  p.  212.  3.  Paraffin  embedding 
methods,  p.  212.  4.  Preliminary  treatment  of  sections,  p.  215.  5.  The  staining  of 
sections  :  A.  Simple  staining,  p.  216  ;  B.  Differential  staining,  p.  217. 

SECTION  I.— FILM  PREPARATIONS. 

Pathological  material  whether  taken  during  life  or  after  death,  from  man 
or  from  one  of  the  lower  animals,  may  be  examined  : 

1.  either  fresh  and  unstained,  or 

2.  after  drying  and  staining. 

1.  Unstained  preparations. 

(a)  Fluids. — Blood,  fluid  exudates  and  pus  may  be  collected  in  a  Pasteur 
pipette  and  ought  to  be  examined  at  once. 

The  examination  of  blood  may  be  described  in  illustration  of  the  method. 
As  soon  as  the  blood  is  removed  from  the  body  a  drop  is  placed  on  a  slide 
and  covered  with  a  cover-glass  ;  the  blood  spreads  out  in  a  thin  layer  between 
the  slide  and  cover-glass,  and  by  pressing  lightly  on  the  latter  the  excess  can 
be  squeezed  out  at  the  edges  and  wiped  away  with  a  piece  of  soft  linen.  In 
this  way  a  very  thin  uniform  layer  is  obtained  and  must  be  examined  imme- 
diately (obj.  D  ;  oc.  2  Zeiss). 

The  slides  and  cover-glasses  must  be  absolutely  clean,  because  dirt  or 
grease  prevents  the  blood  from  spreading  in  a  thin  and  uniform  layer,  and 
renders  satisfactory  examination  of  it  impossible.  It  is  also  essential  that 
the  red  cells  should  not  be  heaped  one  on  another,  as  this  would  mask  the 
presence  of  micro-organisms. 

Should  the  examination  be  very  prolonged  the  edges  of  the  cover-glass 
may  be  luted  with  paraffin,  but  in  the  majority  of  cases  this  is  unnecessary, 
because  the  blood  at  the  edges  of  the  cover-glass,  being  in  contact  with  the 
air,  coagulates  and  thus  affords  sufficient  protection  to  the  central  parts  of 
the  preparation. 

Serous  exudates,  liquid  pus,  etc.,  should  be  treated  in  the  same  way ;  but  if  the 
pus  be  inspissated  it  must  be  treated  as  though  it  were  a  scraping  from  an  organ. 


204  THE   EXAMINATION   OF   MATERIAL 

A  warm  stage  can  be  used  to  maintain  the  preparations  at  the  temperature  of  the 
body  (p.  135). 

(b)  Scrapings  of  organs. — Scrapings  of  the  internal  organs  are  to  be 
collected  in  the  manner  already  described  (Chap.  XL),  and  transferred  with 
a  platinum  loop  to  a  slide  on  which  they  are  rubbed  up  in  a  drop  of  filtered 
water  or,  better,  in  a  drop  of  normal  saline  solution  (water  1000,  NaCl  8, 
filter,  distribute  in  tubes,  sterilize  in  the  autoclave) ;  then  spread  the  material 
with  a  platinum  loop,  cover  with  a  cover-glass,  and  examine  at  once 
(obj.  D  ;  oc.  2  Zeiss). 

2.  Stained  preparations. 

Before  being  stained  fluids  and  scrapings  of  organs  should  be  spread  in  a 
thin  layer  on  a  slide  or  cover-glass,  and  dried  and  fixed  to  preserve  the  form 
of  the  cells  and  to  make  them  adhere  to  the  surface  of  the  glass. 

A.  Preparation  of  films. 

(a)  Fluids. 

The  treatment  of  fluids  such  as  blood,  serous  exudates,  pus,  etc.,  will  first 
be  described. 

1.  Spreading  of  films,     (a)  On  cover-glasses. — 1.  Hold   a  perfectly  clean 
cover-glass  by  one  of  its  angles,  A,  and  place  a  drop   of  the  fluid   to  be 
examined  in  the  centre. 

2.  Cover  with  a  second  cover-glass  laying  the  latter 
across  the  former  as  shown  in  the  figure  (fig.  151). 

3.  Take  hold  of  the  second  cover-glass  at  the  angle 
\   B  B,  opposite  to  A,  and  slide  them  apart  so  that  the 
*         liquid  is  spread  out  in  a  thin,  uniform  layer. 

4.  Allow  the  films  to  dry  either  in  the  air  or  by 
placing  them  on  a  drying  stand  heated  to  40°  or  45°  C. 
(fig.  127,  p.  141). 

(/3)  On  slides. — For  pus,  serous  exudates,  etc.,  slides 
may  be  used  in  a  similar  way  to  cover-glasses :  place 
the  drop  of  fluid  near  one  end  of  the  slide,  lay  another  slide  over  it  and 
then  draw  the  two  slides  apart. 

For  blood  the  following  method  is  better  : 

1.  Take  a  perfectly  clean  slide  and  lightly  touch  the  drop  of  blood  as  it 
oozes  from  the  prick,  taking  care  that  the  blood  is  drawn  up  by  the  slide 
and  not  the  slide  pressed  down  on  to  the  drop. 

2.  Hold  the  slide  in  the  left  hand,  apply  the  edge  of  a  cover-glass  to  the 
drop  of  blood  and  the  latter  will  spread  along  the  edge  of  the  cover-glass  by 
capillary  action.     (The  end  of  a  slide  or  a  visiting  card  or  even  a  small  glass 
stirring  rod  will  serve  equally  as  well  as  a  cover-glass.) 


FIG.  152.— Preparation  of  a  blood  film  on  a  slide. 

3.  Draw  the  cover-glass  slowly  and  without  pressing  upon  it  towards  the 
other  end  of  the  slide.  In  this  way  a  very  thin  and  uniform  layer  of  blood 
is  left  on  the  slide  which  dries  as  fast  as  the  cover-glass  passes  over  it  (fig.  152). 

The  preparation  of  satisfactory  blood-films  requires  a  certain  amount  of  practice, 
so  that  if  the  first  attempts  fail  one  must  not  be  discouraged  ;  remember  always 
that  absolutely  clean  and  flat  slides  and  cover-glasses  are  indispensable. 


SIMPLE   STAINING   OF  FILMS  205 

2.  Fixation.— Several  methods  are  available  for  fixing  films  on  slides  and 
cover-glasses. 

(a)  Heat. — The  slide  or  cover-glass  with  the  film  upwards  is  held  in  a 
pair  of  Cornet's  forceps  (fig.  118,  p.  131)  and  passed  three  times  through  the 
heating  flame  of  a  Bunsen  burner  or  spirit  lamp.  The  shape  of  the  cells  is 
somewhat  distorted  by  this  procedure  and  it  cannot  therefore  be  used  (for 
example)  for  fixing  blood-films. 

(/3)  Alcohol-ether. — Pour  two  or  three  drops  of  alcohol-ether  on  the  cover- 
glass  (p.  141)  and  allow  it  to  dry  in  the  air.  This  method  is  preferable  to  the 
preceding  as  it  preserves  absolutely  the  shape  of  the  cells.  It  is  occasionally 
necessary  to  allow  the  solution  to  act  for  several  minutes. 

(7)  Absolute  alcohol. — In  many  cases  absolute  alcohol  can  be  used  in  place 
of  alcohol-ether  for  fixing  films.  The  technique  is  the  same  as  that  described 
in  the  preceding  paragraph.  With  many  dyes  staining  is  facilitated  by 
allowing  the  alcohol  to  act  for  10-30  minutes. 

(S)  Other  solutions  are  occasionally  used  for  fixing  films,  e.g.  osmic  acid 
vapour,  absolute  methyl  alcohol,  etc.  These  will  be  referred  to  when 
occasion  for  their  use  arises. 

(b)  Scrapings  of  organs. 

Films  of  the  internal  organs  are  prepared  as  follows  : 

1.  Transfer  to  a  slide  with  a  platinum  loop  or  pipette  a  small  piece  of  tissue 
from  the  organ,  and  spread  it  by  rubbing  it  on  the  slide  so  as  to  cover  a 
rectangular  area  about  15-20  mm.  square.     Or  a  piece  of  the  tissue  (liver, 
spleen,  etc.)  may  be  taken  up  in  a  pair  of  dissecting  forceps  and  lightly 
rubbed  over  the  surface  of  the  slide. 

The  film  in  any  case  should  be  thin  and  uniform  and  any  lumps  which 
would  interfere  with  the  application  of  a  cover-glass  must  be  removed. 

2.  Dry  as  above. 

3.  Fix  by  heat  or  with  alcohol-ether. 

Films  of  the  brain  or  spinal  cord  should  always  be  washed  several  times  in  the 
alcohol-ether  mixture  after  fixing  to  remove  fatty  matters,  as  these  would  inter- 
fere with  the  subsequent  staining  processes. 

(c)  Sputum. 

When  the  sputum  is  fluid  it  can  be  treated  as  a  fluid  exudate,  but  should 
it  be  tough  or  inspissated  it  should  be  spread  with  a  platinum  loop  on  a 
slide  ;  it  will  facilitate  the  preparation  of  a  thin  and  uniform  film  if  the  slide 
be  gently  heated  while  the  sputum  is  being  spread.  Dry  and  fix. 

B.  Staining  methods. 

Films  whether  of  fluids  or  scrapings  of  organs  contain  structures  of  two 
different  kinds. 

1.  The  groundwork,   which  is  formed  of  tissues  of  animal  origin — cells 
and  amorphous  elements. 

2.  Bacteria,  which  are  of  vegetable  origin. 
Such  films  may  be  stained  in  one  of  two  ways, 

(a)  With  a  simple  stain  by  which  at  a  single  operation  the  groundwork  and 
the  micro-organisms  are  stained  the  same  colour. 

(b)  With   a  double  slain  by   means   of   which    the   micro-organisms   are 
differentiated  from  the  groundwork  by  being  stained  a  different  colour. 

(a)  Simple  staining. 

A  blood  film  or  a  scraping  of  an  organ  may  be  stained  with  any  of  the  dyes 
described  in  Chapter  VIII. 


206  THE   EXAMINATION   OF   MATERIAL 

The  stain  most  generally  used  is  carbol-thionin  (p.  138).  The  technique 
is  as  follows  : 

A.  Cover-glasses. — 1.  Hold  the  cover-glass  in  a  pair  of  Cornet's  or  Debrand's 
forceps  and  pour  on  to  the  film  sufficient  stain  to  cover  the  surface. 

Allow  the  stain  to  act  for  30-60  seconds. 

2.  Wash  in  distilled  water. 

3.  Mount  the  cover-glass  on  a  slide  film  downwards  in  a  drop  of  water. 
Examine  with  a  TV  objective  and  No.  2  ocular. 

4.  If  the  preparation  be  satisfactory  it  may  be  mounted  permanently,  by 
drying  it  in  the  air  or  gently  heating  it  and  then  mounting  in  Canada  balsam. 

To  sum  up  :    stain,  wash  in  water,  dry,  mount  in  balsam. 

B.  Slides. — Films  made  on  slides  are  stained  in  a  similar  manner,     Hold 
the  slide  in  the  left  hand  or  in  a  pair  of  Debrand's  forceps  ;    flood  the  slide 
with  stain  ;   wash  in  water,  dry  ;   place  a  drop  of  cedar-wood  oil  on  the  film 
and  examine  with  an  immersion  lens.     The  preparation  may  be  mounted  by 
placing  a  drop  of  balsam  on  the  film  and  covering  with  a  cover-glass. 

Dilute  carbol-fuchsin,  the  various  carbol-violet  stains,  Kuhne's  or  Loeffler's  or 
Roux's  blue,  etc.  may  any  of  them  be  used  in  suitable  cases  in  place  of  carbol- 
thionin.  The  particular  stain  which  is  most  useful  for  the  detection  and  study  of 
the  different  species  will  be  referred  to  in  the  chapters  devoted  to  those  species. 

The  disadvantage  of  the  simple  stains  is  that  as  they  stain  the  groundwork 
and  the  organisms  the  same  colour  (fig.  153) ;  the  latter  fail  to  stand  out 


v  £X    'v!%; 


FIG.  153. — Simple  staining. 
Scraping  from  gum  stained  with  dilute  carbol-fuchsin  (oc.  2,  obj.  T\.th.  Zeiss). 

conspicuously,  especially  when  they  are  few  in  number  or  when  the  film  is 
thick.  The  methods  of  differential  staining  are  adopted  to  overcome  these 
defects. 

Examination  of  the  blood. — In  the  case  of  blood-films  the  necessity  for 
double  staining  may  be  avoided  by  getting  rid  of  the  groundwork.  Thus 
if  the  haemoglobin  (which  is  the  only  substance  in  the  red  cells  which  takes 
the  stain)  be  eliminated  there  remains  after  staining  a  colourless  groundwork 
on  which  the  micro-organisms  stand  out  conspicuously.  This  result  may 
be  effected  in  one  of  two  ways  : 

(a)  Gunther's  method. — 1.  Dry  the  film  by  gently  heating  it  and  then 


DIFFERENTIAL   STAINING   OF   FILMS  207 

without  passing  it  through  the  flame  cover  it  with  a  5  per  cent,  solution  of 
acetic  acid,  and  leave  for  30  seconds. 

2.  Expose  to  the  vapour  of  ammonia  for  a  few  seconds. 

3.  Wash  in  water. 

4.  Stain,  wash,  dry  and  mount. 

(/?)  Vincent's  method. — 1.  Dry  the  film  by  gently  heating  it,  and,  without 
fixing,  flood  the  film  with  the  following  solution  : 

5  per  cent,  aqueous  solution  of  carbolic  acid,  6  c.c. 

Saturated  aqueous  solution  of  common  salt.  30     ,, 

Glycerin  (pure),  -         30    „ 

and  allow  it  to  act  for  1-2  minutes. 

2.  Wash  in  water,  stain,  etc. 

(7)  Direct  staining  of  blood-films. — Lastly,  simple  staining  with  Losffler's 
blue  gives  very  good  results  with  blood-films  ;  the  red  cells  are  sharply 
differentiated  from  the  micro-organisms,  the  former  being  stained  pale 
green  and  the  latter  deep  blue.  Carbol-thionin  is  also  useful  in  that  it 
stains  the  nuclei  of  the  leucocytes  and  the  organisms  but  leaves  the  red  cells 
practically  unstained.1 

(b)  Differential  staining. 

In  dealing  with  micro-organisms  which  retain  the  stain  by  Gram's  method 
it  is  easy  to  get  a  double-stained  preparation.  But  when  the  organism 
under  investigation  does  not  stain  by  this  method  more  delicate  processes 
which  often  give  less  satisfactory  results  have  to  be  employed.  Finally,  in 
the  search  for  and  in  the  study  of  certain  organisms,  such  for  example  as  the 
tubercle  and  leprosy  bacilli,  special  methods,  of  which  Ehrlich's  is  a  type, 
have  to  be  adopted.  They  will  be  described  in  the  chapter  devoted  to  the 
tubercle  bacillus. 

A.  Gram's  method  and  its  modifications. 

The  procedure  originally  described  by  Gram  has  undergone  various  modifi- 
cations :  reference  will  be  made  to  the  more  important  of  these.  Meanwhile 
the  beginner  must  be  warned  against  the  danger  of  practising  a  large  number 
of  methods.  The  secret  of  success  lies  in  the  thorough  understanding  of  one 
reliable  procedure  ;  if  this  advice  be  neglected  the  result  may  be  error  and 
failure  and  consequent  discouragement.  The  method  described  under  (/3)  is 
the  one  recommended. 

(a)  Gram's  method. — 1.  Flood  the  slide  or  cover-glass  with  aniline- 
gentian-violet  (p.  139).  Let  the  stain  act  for  2-4  minutes. 

2.  Pour  off  the  stain  and,  without  washing,  flood  the  film  with  Gram's 
iodine  solution.     Let  it  act  for  about  a  minute  until  the  preparation  assumes 
a  blackish  tint. 

3.  Wash  in  distilled  water. 

4.  Decolourize  with  absolute  alcohol  (p.  143)  until  the  film  assumes  a  pale 
grey  tint. 

5.  Wash  in  distilled  water. 

6.  Flood  the  film  with  a  solution  of  eosin  : 

Water-soluble  eosin,         -  1  gram. 

Distilled  water,        -  -       200  c.c. 

Allow  the  eosin  to  remain  on  for  1-2  minutes. 

7.  Wash  in  distilled  water  and  dry. 

8.  If  the  preparation  has  been  made  on  a  slide,  a  drop  of  cedar-wood  oil 

1  In  the  case  of  birds'  blood,  the  nuclei  of  the  red  cells  are  deeply  stained  by  these 
dyes. 


208  THE   EXAMINATION   OF   MATERIAL 

may  be  placed  on  the  film  and  the  preparation  examined  at  once  with  an 
oil-immersion  lens. 

Films  made  on  cover-glasses  should  first  be  examined  in  water,  and  if  satis- 
factory they  can  then  be  mounted  in  balsam  after  drying  and  clearing  in 
clove  oil  and  xylol. 

In  preparations  stained  as  above  the  groundwork  is  pink  and  the  micro- 
organisms violet.  Decolourization  must  be  continued  until  all  traces  of 
violet  have  disappeared  from  the  groundwork  (fig.  154). 


FIG.  154. — Gram's  stain. 
Scraping  from  gum  stained  by  Gram's  method  (oc.  2,  obj.  -n>th,  Zeiss). 

Blood-films  stained  by  Gram's  method  give  very  beautiful  preparations. 
When  dealing  with  the  blood  of  birds  the  action  of  the  alcohol  must  be 
continued  until  all  but  the  nuclei  of  the  red  cells  are  decolourized  and  must 
be  stopped  short  of  complete  decolourization.  so  that  after  counter-staining 
with  eosin  the  protoplasm  of  the  red  cells  is  stained  red  while  the  nuclei  of 
the  red  cells  and  the  micro-organisms  are  stained  violet. 

Note. — Vesuvin  can  be  used  as  a  counter-stain  instead  of  eosin. 

Vesuvin,         -  5  grams. 

Distilled  water,        -  -       100  c.c. 

Micro-organisms  which  retain  the  stain  by  Gram's  method  are  then  stained  deep 
violet  while  gram-negative  organisms  and  the  nuclei  of  the  leucocytes  are  dark 
brown  and  the  protoplasm  of  the  leucocytes  light  brown. 

(ft)  Method  recommended. — 1.  Flood  the  film  with  carbol-gentian-violet 
(p.  138).  Stain  for  about  a  minute. 

2.  Without  washing,  replace  the  violet  with  Gram's  iodine  and  stain  for 
1-2  minutes. 

3.  Wash  in  distilled  water. 

4.  Decolourize  with  absolute  alcohol. 

Instead  of  using  absolute  alcohol  alone  the  process  may  be  hastened  by  washing  first 
with  alcohol  then  with  aniline  oil  and  again  with  alcohol.  But  it  should  be  pointed 
out  that  aniline  oil  is  a  very  powerful  decolourizing  agent  and  should  therefore  only 
be  allowed  to  act  for  a  few  seconds. 

5.  Wash  in  distilled  water. 

6.  Counterstain  with  an  aqueous  solution  of  eosin  as  before. 

7.  Wash,  dry,  mount  and  examine  as  above  (a). 

(y)  Nicolle's  method.— 1.  Stain  with  carbol-gentian-violet  (p.  138)  for 
20-30  seconds. 


DIFFERENTIAL  STAINING   OF  FILMS  209 

2.  Without  washing,  replace  the  violet  by  a  modified  Gram's  iodine  solu- 
tion made  as  follows  : 

Iodine,  1  gram. 

Potassium  iodide,    -  2  grams. 

Distilled  water,        -  -       200  c.c. 

Allow  the  solution  to  act  for  4-6  seconds,  renewing  it  once  or  twice  during 
that  period. 

3.  Wash  in  distilled  water. 

4.  Decolourize  with  an  acetone-alcohol  solution  : 

Absolute  alcohol,     -  5  volumes. 

Acetone,          -  1  volume. 

Decolourization  does  not  take  place  at  once   and  is  only  fully  manifested 
after  washing  in  distilled  water. 

5.  Wash  in  distilled  water. 

6.  Stain  the  ground  work  rapidly  with  an  alcoholic  solution  of  eosin  : 

Saturated  solution  of  eosin  *  in  95  per  cent,  alcohol,    -  1  volume. 

Alcohol  (95  per  cent.),      -  2  volumes. 

7.  Wash,  dry,  mount,  and  examine  as  before. 

(8)  Merieux's  method. — In  the  author's  experience  this  method  has  never 
given  results  equal  to  those  obtained  with  the  methods  already  described. 

1.  Stain  with  carbol-violet  as  in  (y). 

2.  Treat  with  the  following  solution  for  4-6  seconds,  renewing  the  solution  once 
or  twice  during  that  time  : 

Iodine,  1  gram. 

Potassium  iodide,    -  2  grams. 

Saturated  solution  of  eosin  2  in  50  per  cent,  alcohol,  -          -         20  c.c. 
Distilled  water,        -  -       200     „ 

3.  Wash  in  distilled  water. 

4.  Decolourize  in  a  1  in  6  solution  of  acetone  in  alcohol  (vide  supra). 

5.  Wash,  dry,  mount  and  examine. 

(e)  Kuhne's  method. — 1.  Stain  for  several  minutes  in  carbol-blue  (p.  138) 
or  in  ammoniacal  blue  (p.  139). 

2.  Wash  in  distilled  water. 

3.  Treat  with  Gram's  iodine  solution  for  2-3  minutes. 

4.  Wash  in  distilled  water. 

5.  Decolourize  with  a  saturated  solution  of  fluorescein  in  absolute  alcohol. 

6.  When  the  ground-work  no  longer  appears  blue,  wash  in  absolute  alcohol 
then  in  clove  oil  and  xylol,  and  mount  in  balsam. 

The  bacteria  appear  violet  on  a  background  lightly  tinted  with  fluorescein. 

B.  Claudius'  method. 

This  method  as  already  described  on  p.  136  can  be  used  for  staining  smear 
preparations. 

C.  Methods  available  for  staining  organisms  which  are  gram-negative. 

1.  Blood-films. 

In  double  staining  blood-films  containing  gram-negative  organisms  use  is 
made  of  the  property  possessed  by  the  red  cells  of  combining  with  eosin,  and 
also  of  the  marked  selective  affinity  shown  by  bacteria  for  the  basic  aniline 
dyes. 

Note.— The  three  methods  about  to  be  described  are  the  original  methods.  They 
have  undergone  many  improvements  which,  being  specially  adapted  to  work  on 
the  Ifematozoa,  will  be  considered  in  the  chapter  (LVIII.)  dealing  with  these 
organisms. 

1  Alcohol-soluble  eosin.  2  Water-soluble  eosin. 

O 


210  THE   EXAMINATION   OF   MATERIAL 

(a)  Laveran's  method.  Method  recommended. — 1.  Flood  the  film  with 
an  aqueous  solution  of  eosin  (p.  207).  Stain  for  about  a  minute. 

2.  Replace  the  eosin  with  a  saturated  aqueous  solution  of  methylene  blue 
and  stain  for  about  30  seconds. 

3.  Wash  in  distilled  water. 

4.  Dry  and  mount  in  balsam. 

The  red  cells  are  stained  pink  while  the  bacteria  and  the  nuclei  of  the 
white  cells  are  blue.  In  the  blood  of  birds  the  nuclei  of  the  red  cells  are 
also  stained  blue. 

(ft)  Chenzinsky's  method. — 1.  Lay  the  cover-glass,  film  side  downwards, 
in  a  small  ground-glass  covered  glass  dish  containing  a  little  of  the  following 
solution  which  must  have  been  recently  prepared  : 

Saturated  aqueous  solution  of  methylene  blue,   -  -         40  c.c. 

0'5  per  cent,  solution  of  water-soluble  eosin  in  70  per  cent. 

alcohol,        -  20     „ 

Distilled  water,        -  -         40     „ 

Leave  the  film  to  stain  in  the  glass  dish  in  the  warm  incubator  at  37°  C.  for 
3-6  hours. 

2.  Then  wash  the  film  in  distilled  water,  dry,  and  mount  in  balsam. 

(y)  Romanowsky's  method. — 1.  After  drying  and  fixing  in  the  flame, 
place  the  film  in  a  drying  oven  at  105°-110°  C.  for  about  an  hour. 

2.  Then  immerse  the  cover-glass  in  the  following  staining  solution  which 
must  be  newly  made  up  and  not  filtered  : 

Saturated  aqueous  solution  of  Hochst's  medicinal  methy- 
lene blue,    -  2  parts. 
1  per  cent,  aqueous  solution  of  eosin  A.G.  (Hochst),    -  5       „ 

Stain  for  2-10  hours. 

3.  Wash  in  distilled  water. 

4.  Dry,  and  mount  in  balsam. 

2.  Films  of  pus,  etc. 
(a)  Ktihne's  method. — 1.  Stain  for  a  few  minutes  with  carbol-blue  (p.  138). 

2.  Wash  in  water. 

3.  Wash  in  dilute  hydrochloric  acid  until  the  film   assumes  a  pale  blue 
colour  (this  is  rather  a  delicate  proceeding  and  the  time  required  will  vary 
with  the  thickness  of  the  film). 

Dilute  hydrochloric  acid. 

Pure  hydrochloric  acid,    -  1  c.c. 

Distilled  water,        -  -         1000     „ 

4.  Remove  the  excess  of  acid  by  washing  in  an  alkaline  lithia  solution. 

Saturated  aqueous  solution  of  carbonate  of  lithia,        -          -  5  c.c. 

Distilled  water,        -  100     „ 

5.  Wash  well  in  water. 

6.  Dry,  clear  in  clove  oil  and  xylol,  and  mount  in  balsam.     The  ground- 
work is  stained  pale  blue  and  the  micro-organisms  deep  blue. 

(ft)  Nicolle's  method.  Method  recommended. — 1.  Stain  for  a  few  minutes 
in  carbol-blue. 

2.  Wash  in  water. 

3.  Treat  for  2  or  3  seconds  with  a  few  drops  of  the  following  solution  : 

Pure  tannin,  -  ...         .  10  grams. 

Distilled  water,        -  100       „ 

4.  Wash  in  water. 

5.  Treat  rapidly  with  absolute  alcohol,  clove  oil  and  xylol,  and  mount 
in  balsam. 


SECTION  CUTTING  211 

The  ground-work  is  stained  very  pale  violet-blue  and  the  organisms  deep 
blue. 

SECTION  II.^HISTOLOGICAL  PREPARATIONS. 

For  the  demonstration  of  micro-organisms  in  situ  in  tissues  very  thin 
sections  (0'05  mm.)  must  be  cut.  Hand-cut  sections  are  not  sufficiently 
thin  for  purposes  of  bacteriological  investigation,  so  that  the  tissue  must 
be  cut  with  a  microtome,  which  involves  the  embedding  of  the  tissue  first  of 
all  in  some  suitable  material. 

The  materials  ordinarily  used  in  histology  for  embedding  tissues  (gum, 
wax,  soap,  celloidin  and  collodion)  do  not  lend  themselves  to  the  cutting  of 
very  thin  sections,  so  that  for  bacteriological  purposes  the  tissue  is  either 
frozen  or  embedded  in  paraffin. 

1.   Instruments. 

Microtomes. — Most  of  the  mechanically- worked  microtomes  are  suitable 
for  cutting  the  thin  sections  required  in  bacteriological  work.  For  paraffin 
sections,  Minot's,  Radais'  and  the  Cambridge  "  rocking  "  microtome  (fig.  155) 
are  among  those  in  most  frequent  use. 


FIG.  155. — Cambridge  "  rocking  "  microtome. 

It  will  be  unnecessary  here  to  discuss  the  construction  of  the  different 
forms  of  microtome  and  the  method  of  working  them,  for  a  careful  examina- 
tion of  the  instrument  itself  will  be  of  far  more  assistance  than  any  detailed 
description. 

It  will  suffice  to  say  that  microtomes  being  instruments  of  precision  must 
be  carefully  handled  ;  that  they  must  be  cleaned  every  time  after  use,  and  be 
protected  from  dust  and  damp  by  being  kept  under  a  bell  jar  or  in  a  wooden 
box. 

Microtome  razors. — A  good  razor  is  indispensable  for  the  cutting  of  satis- 
factory sections.  One  surface  of  the  razor  must  be  flat  (the  one  in  contact 
with  the  paraffin  block).  The  cutting  edge  must  be  sufficiently  sharp  to 
sever  an  hair  held  between  the  finger  and  thumb  or  one  of  the  fine  hairs  on 
the  back  of  the  hand. 

Always  strop  the  razor  before  using  it,  first  on  the  prepared  surface  of 
the  strop  and  then  on  the  dry  surface,  remembering  to  strop  with  the  back 
foremost  and  to  pass  from  heel  to  tip,  stropping  each  side  of  the  razor 
alternately. 

It  is  useful  also  to  ensure  satisfactory  results  and  to  avoid  having  to  send 
it  frequently  to  the  instrument-maker  to  know  how  to  sharpen  a  razor  on  a 
stone.  The  razor  must  be  passed  with  the  edge  foremost  from  heel  to  tip  ; 


212  HISTOLOGICAL  PREPARATIONS 

the  stone  should  not  be  oiled,  but  simply  moistened  with  a  little  water  or 

better  still  with  the  following  solution  : 

Distilled  water,        -  -         50  c.c. 

Alcohol  (95  per  cent.),      -  -         50     „ 

Glycerin,         .....  -         50     „ 

After  use  the  razor  should  be  dried  on  a  piece  of  soft  rag,  lightly  stropped, 
and  returned  to  its  case. 

To  cut  sections  embedded  in  paraffin  the  blade  of  the  razor  should  be  dry 
and  be  placed  obliquely  to  the  tissue.  The  sections,  as  they  are  cut,  should 
be  picked  up  from  the  razor  with  a  pair  of  fine  forceps  or  a  piece  of  silk  paper, 
never  with  a  needle  or  scalpel  or  other  similar  instrument  which  might 
damage  the  cutting  edge  of  the  razor. 

2.  Freezing  methods. 

Though  frozen  tissues  cannot  be  cut  so  thin  as  tissues  embedded  in  paraffin, 
the  freezing  method  has  the  advantage  that  sections  can  be  cut  in  a  very  short 
time,  and  can  be  stained  in  a  variety  of  ways ;  and  hence  is  of  particular 
value  for  purposes  of  rapid  diagnosis. 

Only  tissues  which  have  been  previously  fixed  should  be  cut  by  the  freezing 
method.  Formalin  (10  per  cent.)  is  perhaps  the  best  for  the  purpose  (p.  189), 
as  tissues  can  be  frozen  without  any  further  treatment.  Tissues  fixed  by 
other  methods  should  be  washed  and  then  put  in  formalin  for  a  few  hours. 
Tissues  for  frozen  sections  should  not  be  more  than  5-6  mm.  thick. 

Microtomes. — The  simplest  type  for  frozen  sections  is  a  rocking  microtome 
or  a  Minot.  Place  the  tissue  wet  with  formalin  on  the  carrier  of  the  micro- 
tome and  direct  a  jet  of  methyl  chloride  on  to  it  until  it  is  firmly  frozen  to 
the  carrier,  then  adjust  the  latter  to  the  microtome  and  cut  the  sections. 

Of  microtomes  specially  arranged  for  cutting  frozen  sections  the  best  are 
those  of  Becker  and  Miller,  in  which  the  tissue  is  frozen  by  the  decompression 
of  liquid  carbonic  acid.  The  tissue  is  placed  in  an  hollow  carrier  connected 
by  an  iron  tube  to  a  cylinder  of  car"bonic  acid,  and  when  arranged  in  place 
on  the  microtome  is  frozen  by  simply  turning  on  the  tap  of  the  cylinder. 
When  the  tissue  is  frozen  the  gas  is  turned  off  and  the  sections  cut.  If  the 
sections  show  a  tendency  to  tear,  it  is  because  the  tissue  has  been  frozen 
too  hard,  in  which  case  it  must  be  left  for  a  few  seconds. 

Transfer  the  sections  to  ordinary  water  in  which  they  will  uncurl  ;  when 
uncurled  they  are  ready  for  staining. 

3.  Paraffin  embedding  methods. 

A.  Xylol  method.  Method  recommended. — The  pieces  of  tissue  after  being 
fixed  in  the  manner  described  in  Chapter  XL  are  treated  as  follows: 

1.  Dehydrate  carefully  in  absolute    alcohol  or  acetone  for  24  hours  or 
thereabouts. 

2.  Transfer  to  xylol. 

Very  small  pieces  (1-3  mm.)  for         -  -         30-60  minutes. 

Small  pieces  (3-5  mm.),  -  2     hours. 

Medium-sized  pieces  (5-10  mm.),       -  3-4        „ 

Thick  pieces  (10  mm.  or  more).  4-5        ,, 

In  the  case  of  the  last  it  is  as  well  to  change  the  xylol  once  or  twice  to  make  quite 
sure  that  all  traces  of  alcohol  will  be  removed. 

3.  After  dehydrating,  transfer  to  a  mixture  of  xylol  and  paraffin  melting 
at  35°  C.     Such  a  mixture  can  be  made  as  follows  : 

Paraffin1  (melting  point  50°  C.),        ....  10-15  grams. 

Xylol,     -          -  30  c.c. 

1  For  embedding,  the  paraffin  sold  by  Dumaige  of  Paris  is  recommended. 


PARAFFIN  EMBEDDING  213 

The  tissue  should  be  placed  in  the  mixture  in  a  well-stoppered  bottle  and 
be  kept  in  the  warm  incubator  (37°-38°  C.)  for  from  1-6  hours  according  to 
the  thickness  of  the  block. 

4.  After  passing  through  the  xylol-paraffin  bath  transfer  to  an  open  flask 
or  tube  containing  paraffin  melting  at  50°  C.  and  heated  to  52°-53°  C.  (the 


FIG.  156. — Paraffin  oven. 

temperature  must  never  reach  55°  C.)  in  a  paraffin  oven  (fig.  156)  for  £-4 
hours  according  to  the  thickness  of  the  tissue. 

Very  thin  pieces,     -  30  minutes. 

Thin  pieces,    -  1-2  hours. 

Medium-sized  pieces,        .......          2-3       „ 

Thick  pieces,  3-5       „ 

5.  The  tissue  is  now  ready  to  be  embedded.  Melt  some  paraffin  (melting 
point  50°,  52°  or  55°  C.)  in  a  porcelain  capsule.  (For  sections  for  bacterio- 
logical examination  paraffin  melting  at  52°  C.  is,  generally  speaking,  the 
best,  but  if  the  weather  is  very  warm  paraffin  melting  at  55°  C.  may  be 
preferred.)  After  the  paraffin  has  been  melted  allow  it  to  cool  until  a  pellicle 
forms  on  the  surface. 

While  the  paraffin  is  melting  select  a  mould  and  cover  the  bottom  with  a 
thin  layer  of  the  melted  paraffin,  and  as  soon  as  it  has  begun  to  set  (a  few 
seconds  is  sufficient)  place  the  tissue,  which  may  be  conveniently  held  with  a 
lightly  heated  needle,  on  the  surface,  taking  care  that  it  is  placed  in  a  good 
position  and  suitably  orientated  ;  then  fill  up  the  mould  with  melted  paraffin, 


214  HISTOLOGICAL  PREPARATIONS 

being  careful  that  the  tissue  is  embedded  to  a  depth  of  several  millimetres 
to  allow  for  the  contraction  which  will  take  place  during  cooling. 

As  soon  as  the  paraffin  has  set  sufficiently  to  hold  the  tissue  the  needle 
which  was  used  to  retain  the  latter  in  position  should  be  taken  away.  The 
paraffin  should  be  cooled  rapidly  by  plunging  the  mould  into  cold  water, 
being  careful  first  to  moisten  the  bottom  and  not  to  immerse  the  mould  com- 
pletely before  the  paraffin  has  cooled  sufficiently  to  allow  of  the  formation  of 
a  crust  on  the  surface,  otherwise  of  course  the  water  would  penetrate  into 
the  paraffin  and  destroy  the  homogeneity  of  the  mass. 

6.  When  the  paraffin  is  firmly  set,  take  it  out  of  the  mould  and  the  tissue 
is  ready  for  cutting. 

Paraffin  moulds. — 1.  The  simplest  mould  is  one  made  out  of  paper  in  the 
following  manner  :  Select  a  cork  which  loosely  fits  the  carrier  on  the  micro- 
tome, and  roll  round  it  a  strip  of  filter  paper — which  may  be  fastened  by 
pinning  it  to  the  cork — so  as  to  form  an  hollow  cylinder  2  or  3  cm.  deep,  the 
bottom  being  formed  by  the  upper  surface  of  the  cork.  This  surface  may, 
with  advantage,  be  scored  with  a  few  small  grooves  cut  with  a  scalpel  to 
ensure  the  paraffin  adhering  more  firmly  to  it.  Oil  the  inner  surface  of  the 
paper  with  a  brush  avoiding  the  surface  of  the  cork  at  the  bottom  of  the 
cylinder. 

Pour  the  melted  paraffin  into  this  cylinder  and  when  it  has  set  take  out 
the  pin  and  unroll  the  paper  ;  the  paraffin  with  the  tissue  embedded  in  it 
will  remain  attached  to  the  cork.  Trim  up  the  surface  of  the  paraffin  and 
fix  the  cork  into  the  carrier  of  the  microtome.  The  block  is  then  ready  for 
cutting. 

2.  The  lead  capsules  used  for  covering  the  corks  of  bottles  serve  the  same 
purpose  excellently.    When  the  paraffin  has  set  the  capsule  is  torn  off,  leaving 
a  solid  block  of  paraffin  which  can  be  trimmed  up  at  leisure  with  a  slightly 
heated  scalpel.     Blocks  cast  in  such  a  mould  can  be  easily  fitted  to  the  carrier 
of  the  microtome.     In  using  a  Minot  microtome  it  is  only  necessary  to  heat 
gently  the  grooved  metal  carrier  and  to  press  the  lower  surface  of  the  paraffin 
block  lightly  against  it.     To  fix  the  block  to  the  wooden  cube  or  cylinder  used 
with  other  microtomes  apply  the  blade  of  a  lightly  heated  scalpel  to  the  lower 
surface  of  the  block,  and  while  the  paraffin  is  still  soft  press  it  on  to  the  wood 
block  ;   or  if  preferred  a  little  melted  paraffin  may  be  poured  on  to  the  latter 
and  the  paraffin  block  pressed  on  to  it. 

In  the  same  way  small  cardboard  or  wooden  boxes,  cover-glass  boxes  for 
example,  make  very  good  moulds  ;  these  must  be  painted  on  the  inside  with 
glycerin  or  oil  to  prevent  the  paraffin  adhering  to  the  sides. 

3.  By  using  Leuckart's  moulds  blocks  of  various  sizes  with  perfectly  smooth 
and  parallel  sides  are  obtained.     These  moulds  consist  of  two  pieces  of  brass, 
which  can  be  placed  together  in  such  a  way  that  they  form  a  rectangular 
box  (fig.  157).     The  surfaces  of  the  two  pieces  of  metal  are  smeared  with 


FIG.  157.— Paraffin  moulds. 


glycerin  and  laid  on  a  piece  of  glass  which  has  also  been  smeared  with  glycerin 
and  they  are  then  arranged  so  as  to  form  a  box  of  the  size  required.  The 
melted  paraffin  is  poured  into  the  box  and  when  it  has  set  the  two  pieces 


PRELIMINARY   TREATMENT  215 

•of  metal  are  pushed  apart  and  the  paraffin  with  the   tissue   embedded   is 
free. 

B.  Toluene  method. — The  technique  is  exactly  the  same  as  when  using 
xylol  except  that  toluene  is  substituted  for  xylol. 

C.  Ether  method. — 1.  When  the  tissue  is  taken  out  of  absolute  alcohol  it 
is  transferred  to  alcohol-ether  for  from  30  minutes  to  6  hours  according  to 
the  size  of  the  tissue. 

2.  The  tissue  is  then  immersed  in  pure  ether  for  at  least  as  long  as  it  was 
in  the  alcohol-ether  mixture. 

3.  It  is  then  transferred  to  an  hermetically  sealed  flask  containing  ether 
saturated  with  paraffin  melting  at  50°  C.  and  placed  in  the  warm  incubator 
at  37°-38°  C.  (see  under  xylol  for  duration  of  treatment). 

4.  The  block  is  now  immersed  in  paraffin  melting  at  50°  C.  and  embedded 
in  the  manner  described  under  xylol. 

4.  Preliminary  treatment  of  sections. 

Before  sections  can  be  stained  the  paraffin  which  has  penetrated    the 
interstices  of  the  tissue  must  be  removed. 

A.  Method  recommended. — 1.  As  soon  as  they  are  cut  the -sections  are 
placed  in  a  ground-glass  stoppered  vessel  containing  ether  which   rapidly 
dissolves   the   paraffin.     The  length  of  time  required  will  vary 

from  several  minutes  to  a  few  hours  according  to  the  size  and 
number  of  the  sections  treated. 

2.  When  all  the  paraffin  has  dissolved  the  sections  are  trans- 
ferred with  a  platinum  or  nickel  spatula  (fig.  158)  to  a  second 
bath  containing  absolute  alcohol. 

3.  After  being  in  absolute  alcohol  for  a  few  minutes  the  sections 
are  transferred  one  by  one  with  a  spatula  to  a  glass  dish  full  of 
distilled  water.     As  soon  as  they  come  in  contact  with  the  water 
the  sections  spin  round  and  round  very  rapidly  and  at  the  same 
time  unroll  and  spread  themselves  flat. 

If  the  sections  are  very  thin  and  fragile  this  gyratory  movement 
may  tear  them  and  render  them  useless,  so  that  it  is  better  to  pass 
such  sections  from  absolute  alcohol  to  70  per  cent,  then  to  40  per  cent, 
alcohol  before  placing  them  in  distilled  water. 

4.  To  transfer  a  section  to  a  slide,  dip  the  slide  obliquely  into 
the  water  and  beneath  one  of  the  sections,  then  fixing  the  section 
with  a  needle  raise  the  slide  and  gently  draw  it  out  of  the  water, 
holding  the  section  with  the  needle  about  the  centre  of  the  slide 
on  which  it  will  spread  out.     Blot  up  the  excess  of  water  with  a 
cigarette  paper  or  a  piece  of  silk  paper  (which  should  be  kept 
ready  cut  up  into  small  rectangular  pieces,  and  not  torn  off  as 
required  since  the  rough  edges  might  pick  up  the  section  from 

the  slide)  and  the  section  is  now  ready  for  staining.  FlG.  158  _ 

_         Section  lifter. 

B.  Albumin  fixation. — The  method  just  described  is  the  simplest 

and,  in  the  hands  of  those  used  to  the  work,  applicable  to  the  majority'  of 
cases.  But  when  the  sections  are  very  delicate — sections  of  lung,  for  instance, 
—there  is  a  risk  that  they  may  be  torn  during  the  various  manipulations. 
In  such  a  case  it  is  invariably  necessary  to  fix  the  section  on  the  slide 
immediately  it  is  cut.  The  fixative  generally  used  in  bacteriology  is  Mayer's 
albumin. 
Mayer's  albumin. — Beat  up  the  white  of  two  eggs  into  a  snow,  leave  them 


216  HISTOLOGICAL  PREPARATIONS 

to  stand,  then  filter  through  filter  paper  and  add  an  equal  volume  of  glycerin 
to  the  clear  filtrate.  Add  a  little  piece  of  camphor  or  thymol  as  a  preserva- 
tive and  keep  in  a  well-stoppered  bottle.  Before  using  the  solution  shake  the 
bottle  well  to  ensure  the  mixture  being  homogeneous. 

Method  of  use. — Place  a  drop  of  the  albumin  on  the  slide  and  spread  it  in 
a  very  thin  layer  with  the  tip  of  the  index  finger.  Transfer  the  section  with 
a  spatula  direct  to  the  prepared  slide,  carefully  spread  it  out  with  a  fine 
brush  so  that  there  are  no  folds  and  press  it  lightly  to  make  it  adhere  to 
the  albumin. 

Should  there  be  any  difficulty  in  getting  the  sections  to  spread,  a  drop  of  water 
may  be  placed  on  the  slide  already  smeared  with  the  albumin  mixture  and  the 
section  laid  on  the  drop  of  water.  The  slide  is  then  gently  heated  on  the  drying 
stage  (fig.  127,  p.  141)  until  the  section  has  spread  evenly,  the  excess  of  water  is 
then  taken  up  with  a  piece  of  silk  paper  and  the  process  continued  as  below. 

Heat  the  under  side  of  the  slide  very  lightly  over  the  pilot  flame  of  a  Bunsen 
and  in  a  few  seconds  the  section  will  have  adhered  to  the  surface  of  the  glass. 
The  section  is  now  treated  with  xylol  and  then  with  absolute  alcohol  to 
remove  the  paraffin,  after  which  it  is  ready  for  staining. 

Note. — The  albumin-fixation  method  has  the  disadvantage  of  not  being  universally 
applicable:  it  cannot,  for  instance,  be  used  with  alkaline  solutions,  Orth's  picro- 
carmine,  etc.,  -which  dissolve  albumin. 

5.  The  staining1  of  sections. 

In  order  to  render  the  detection  of  micro-organisms  as  easy  as  possible  and 
to  facilitate  their  study,  it  is  desirable  that  they  should  be  stained  a  different 
colour  from  the  tissue  in  which  they  are  contained ;  hence  it  is  best  to  use 
either  a  double  or  triple  staining  method.  Unfortunately  such  methods  are 
of  little  use  when  dealing  with  an  organism  which  is  gram-negative  and 
which  does  not  stain  either  by  Ehrlich's  or  Ziehl-Neelsen's  method.  In 
such  a  case  it  is  sometimes  not  possible  to  differentiate  further  than  by  staining 
with  a  simple  stain  in  such  a  way  that  the  background  (the  animal  tissue)  is 
only  lightly  stained  while  the  bacteria  (the  vegetable  tissue)  are  stained  much 
more  deeply.  Recently,  however,  methods  of  double  staining  applicable  to 
gram-negative  organisms  have  been  devised  and  two  of  these  will  be  described. 

The  description  of  Ehrlich's  and  Ziehl-Neelsen's  methods  will  be  deferred 
to  the  chapter  on  tuberculosis. 

A.  Simple  staining. 

Methods  applicable  to  most  organisms. 

(a)  Weigert's  method. — 1.  Cover  the  section  with  a  few  drops  of  aniline- 
gentian-violet  (p.  139).  Allow  the  stain  to  act  for  30  minutes  or  so  and  then 
blot  up  the  excess. 

2.  Immerse  the  section  for  a  few  seconds  in  a  vessel  containing  a  0'5  per 
cent,  aqueous  solution  of  acetic  acid. 

3.  Wash  carefully  in  distilled  water  and  blot  up  the  excess. 

4.  Dehydrate  very  rapidly  in  absolute  alcohol. 

5.  Clear  in  clove  oil  then  in  xylol. 

6.  Mount  in  Canada  balsam. 

(ft)  Loeffler's  method.— The  stains  used  are  Loeffler's  alkaline  blue  (p.  139) 
(15  minutes)  or  Ziehl's  fuchsin  (p.  138)  (5  or  6  minutes).  The  technique  is 
otherwise  the  same  as  in  the  preceding  method. 

(7)  Kiihne's  method  A.— 1.  Stain  for  15  minutes  in  carbol-blue  or 
ammoniacal  blue  (pp.  138  and  139). 

2.  Transfer  to  distilled  water. 


SIMPLE  STAINING  217 

3.  Treat  for  a  few  seconds  with  dilute  hydrochloric  acid  (1-1000). 

4.  Transfer  rapidly  to  lithia  solution  (p.  210). 

5.  Wash  again  carefully  in  distilled  water.     Blot  up  the  excess  of  water 
and  leave  the  section  exposed  to  the  air  until  it  is  nearly  dry. 

6.  Dehydrate  as  rapidly  as  possible  in  absolute  alcohol. 

7.  Clear  in  clove  oil  and  xylol. 

8.  Mount  in  Canada  balsam. 

(5)  Kuhne's  method  B. — This  method  is  not  recommended.  It  is  very 
tedious  and  only  stains  a  few  species  of  micro-organisms. 

1.  Stain  for  about  30  minutes  in  carbol-blue. 

2.  Wash  in  distilled  water. 

3.  Treat  with  dilute  hydrochloric  acid  (1-1000)  until  the  tissue  is  pale  blue. 

4.  Wash  in  lithia  solution  (p.  210). 

5.  Wash  for  several  minutes  in  distilled  water  and  blot  up  the  excess. 

6.  Dehydrate  very  rapidly  in  absolute  alcohol  lightly  tinted  with  methylene  blue. 

7.  Pour  off  the  alcohol  and  treat  with  aniline  oil  similarly  tinted  with  blue  for 
about  2  minutes. 

8.  Replace  the  tinted  aniline  oil  with  ordinary  aniline  oil  for  about  2  minutes. 

9.  Clear  with  clove  oil  and  then  with  two  lots  of  xylol  to  ensure  the  removal  of 
all  traces  of  aniline  oil. 

10.  Mount  in  Canada  balsam. 

(e)  Staining  with  thionin.     Method  recommended.— 1.  Stain  with  carbol- 

thionin  (p.  138)  for  several  minutes. 

2.  Wash  in  distilled  water  and  blot  up  the  excess. 

3.  Dehydrate  very  rapidly  in  absolute  alcohol. 

4.  Clear  in  clove  oil  and  xylol. 

5.  Mount  in  Canada  balsam. 

(0  Gram's  method  for  the  typhoid  bacillus. — 1.  Stain  for  a  few  hours  in 
aniline-gentian-violet  (p.  139). 

2.  Wash  the  section  in  distilled  water. 

3.  Transfer  for  1  minute  to  a  1  per  cent,  solution  of  hydrochloric  acid. 

4.  Wash  carefully  in  distilled  water  :  blot  up  the  excess. 

5.  Dehydrate  very  rapidly  in  absolute  alcohol. 

6.  Clear  in  clove  oil  and  xylol. 

7.  Mount  in  Canada  balsam. 

By  this  method  the  bacilli  alone  are  stained. 

(rj)  Nicolle's  tannin  method.  Method  recommended. — 1.  Stain  the  section 
for  2  or  3  minutes  in  Lceffler's  or  Kuhne's  blue. 

2.  Wash  in  distilled  water. 

3.  Treat  for  a  few  seconds  in  a  10  per  cent,  aqueous  solution  of  tannin. 

4.  Wash  in  distilled  water  and  blot  up  the  excess. 

5.  Dehydrate  rapidly  in  absolute  alcohol. 

6.  Clear  in  clove  oil  and  xylol. 

7.  Mount  in  Canada  balsam. 

B.  Differential  staining. 
1.    Methods  applicable  to  gram-positive  organisms. 

To  demonstrate  the  presence  of  gram-positive  organisms  in  a  tissue  in 
which  they  are  present  the  background  (the  animal  tissue)  is  first  stained  with 
an  acid  dye  which  has  but  little  affinity  for  micro-organisms,  then  by  Gram's 
method.  The  bacteria  being  the  only  structures  stained  violet  stand  out 
sharply  from  the  other  tissues. 

The  background  may  be  stained  with  one  of  several  dyes. 

For  double  staining,  eosin,  fluorescein,  carmine  (Orth's),  vesuvin,  Boehmer's 
hsematoxylin,  aurantia,  hsematein,  etc.,  are  used. 


218  HISTOLOGICAL  PREPARATIONS 

For  triple  staining  a  selective  dye  is  chosen  which  will  stain  the  various 
tissues  different  colours.  This  method  of  staining  enables  the  lesions  produced 
by  the  organisms  to  be  studied.  The  stains  ordinarily  used  are  Orth's  picro- 
carmine  or  haematoxylin  in  conjunction  with  aurantia  or  eosin.  The  following 
are  the  formulae  most  commonly  in  use : 

STAINING  SOLUTIONS. 

Dilute  aqueous  solution  of  eosin. 

Water-soluble  eosin,          -  0'50  gram. 

Distilled  water,        -  -       300        c.c. 

Filter. 

Solutions  of  fluorescein,  aurantia,  vesuvin  (0'5  per  cent.),  etc.,  are  prepared  in  a  similar 
manner. 

Bcehmer's  hcematoxylin. 

Make  up  the  two  following  solutions  : 

(a)  Hsematoxylin  crystals,      -          -  1  gram. 

Absolute  alcohol,     -         -  -         10  c.c. 

Pour  the  solution  into  a  well-stoppered  bottle. 
(6)  Potash  alum,  -----  -         20  grams. 

Distilled  water,        ...  -       200  c.c. 

Dissolve  in  the  warm  and  filter  when  cool. 

Allow  to  stand  for  24  hours  and  then  mix  the  two  solutions  a  and  b  ;  leave 
the  mixture  exposed  to  the  air  for  a  week,  then  store  in  a  well-stoppered 
bottle  and  filter  immediately  before  use. 

Hcematein. 

Prepare  the  two  following  solutions  : 
(a)  Hsematein,      -  1  gram. 

Absolute  alcohol,     -  -         50  c.c. 

(6)  Potash  alum,  -         -  -         50  grams. 

Distilled  water,        -  -     1000  c.c. 

The  potash  alum  is  dissolved  in  the  warm  and  added  immediately  to  the 
haematein  solution.  Let  the  mixture  cool  in  the  air  and  then  filter. 

Orth's  carmine. 

Saturated  aqueous  solution  of  carbonate  of  lithia,        -          -     100        c.c. 
Carmine  No.  40,      -  -         2*50  grams. 

Dissolve  by  trituration  in  a  mortar  in  the  cold. 

Orth's  alcohol  carmine. 

Orth's  carmine,        -  5  volumes. 

95  per  cent,  alcohol,         .......  1  volume. 

Mix. 

The  latter  solution  only  should  be  used  for  staining  sections  fixed  with 
Mayer's  albumin,  as  the  former — the  non-alcoholic  solution — dissolves 
albumin. 

Orth's  picrocarmine. 
Mix. 

Orth's  carmine,        -  1  volume. 

Saturated  aqueous  solution  of  picric  acid,  -  1-2  volumes. 

After  staining  in  the  picrocarmine  solution,  the  sections  should  be  trans- 
ferred to  the  following  fixing  solution  : 

Absolute  alcohol,     -  -      70        c.c. 

Saturated  aqueous  solution  of  picric  acid,  -          -          -          -      30          ,, 
Pure  hydrochloric  acid,    -  -        0'50  gram. 


DIFFERENTIAL   STAINING  219 

(i)  Double  staining. 

A.  Method  recommended. — 1.  Treat  the  section  for  about  30  seconds  with 
the  dilute  solution  of  eosin  (p.  218)  until  it  acquires  a  pink  colour. 

2.  Wash  in  distilled  water. 

3.  Stain  the  section  on  the  slide  for  about  30  seconds  with  carbol-gentian- 
violet  or  carbol-crystal- violet  (p.  138).     It  now  assumes  a  violet  colour. 

4.  Pour  off  the  violet  and  treat  the  section  with  Gram's   iodine  for  30 
seconds  or  so,  renewing  the  solution  two  or  three  times  until  the  section  is 
black.     Wash  in  distilled  water. 

5.  Wash  with  absolute  alcohol  (or  absolute  alcohol  and  aniline  oil)  until 
the  pink  colour  of  the  ground-work  reappears. 

6.  Clear  with  clove  oil  and  xylol. 

7.  Mount  in  balsam. 

The  background  is  stained  pink  and  those  organisms  which  retain   the 
stain  by  Gram's  method  are  stained  violet. 

B.  Kiihne's  method. — 1.  Stain  the  section  for  5-15   minutes  in  Kiihne's 
blue  or  ammoniacal  blue. 

2.  Wash  in  distilled  water. 

3.  Treat  with  Gram's  solution  for  2  or  3  minutes. 

4.  Wash  in  distilled  water. 

5.  Decolourize  in  a  saturated  solution  of  fluorescein  in  absolute  alcohol. 

6.  Treat  with  pure  absolute  alcohol,  clove  oil  and  xylol. 

7.  Mount  in  balsam. 

Bacteria  are  stained   blue  while  the  ground-work  is  faintly  stained  with 
fluorescein. 

(ii)  Triple  staining. 

A.  Method   recommended. — 1.  Stain   for   about   5   minutes   with    Orth's 
picrocarmine. 

2.  Pour  off  the  stain  and  fix  in  the  fixing  solution  for  about  30  seconds. 

3.  Wash  in  distilled  water. 

4.  Stain  with  carbol-gentian-violet  or  carbol-crystal-violet  for  30  seconds. 

5.  Replace  the  stain  with  Gram's  solution  for  30  seconds.     Wash  in  dis- 
tilled water. 

6.  Decolourize  in  absolute  alcohol  or  absolute  alcohol  and  aniline  oil. 

7.  Treat  the  section  in  turn  with  absolute  alcohol  slightly  tinted  with  picric 
acid,  clove  oil  and  xylol. 

8.  Mount  in  balsam. 

B.  Nicolle's  method. — This  method  is  applicable  to  sections  fixed  on  the 
slide  with  Mayer's  albumin. 

1.  Stain  with  Orth's  alcohol-carmine  for  15  minutes. 

2.  Wash  in  distilled  water. 

3.  Stain  in  carbol-gentian-violet  (p.  138)  for  6  seconds. 

4.  Substitute  Gram's  strong  solution  (p.  209)  for  the  gentian-violet  and 
treat  for  4  or  6  seconds,  renewing  the  solution  twice  during  the  process. 

5.  Decolourize  with  alcohol-acetone  (1  to  3). 

6.  Transfer  to  picric  acid  in  absolute  alcohol  for  a  second  or  two. 

7.  Clear  in  clove  oil  and  xylol. 

8.  Mount  in  balsam. 

C.  Claudius'  method. — 1.  Fix  the  section  on  the  slide  with  Mayer's  albumin. 

2.  Stain  for  10-15  minutes  in  Orth's  alcohol-carmine. 

3.  Wash  in  distilled  water. 

4.  Stain  for  2  minutes  in  a  1  per  cent,  aqueous  solution  of  methyl  violet 
or  in  carbol-gentian-violet. 


220  HISTOLOGICAL   PREPARATIONS 

5.  Treat  for  2  minutes  with  picric  acid  solution  (p.  144). 

6.  Blot  up  the  picric  solution  carefully  with  filter  paper  and  pour  a  large 
drop  of  chloroform  over  the  section.     Blot  up  the  chloroform  with  filter  paper 
and  replace  it  with  a  drop  of  clove  oil  and  repeat  the  process  until  the  section 
assumes  a  pink  colour. 

7.  Clear  in  xylol  and  mount  in  balsam. 

2.    Methods  applicable  to  organisms  in  general. 

A.  Foa's  method. — This  method  is  particularly  useful  for  the  detection  of 
the  typhoid  bacillus.     It  depends  upon  the  use  of  a  mixture  of  methyl-green 
and  pyronin  (Pappenheim's  solution). 

When  this  method  of  staining  is  to  be  used  the  tissue  should  not  be  fixed  in  alcohol 
but  in  the  following  solution  : 

Perchloride  of  mercury, 2  grams. 

Mailer's  fluid,    -  -      100  c.c. 

Leave  the  tissue  in  this  solution  for  24-48  hours  ;    wash  in  water  for  2  hours  ; 
harden  in  alcohol  (p.  188)  and  embed  in  paraffin. 

1.  Stain  the  sections  for  5  minutes  in  the  following  mixture  : 

Saturated  aqueous  solution  of  methyl-green  (Griibler),  3-4  volumes, 

pyronin,       -  1-2 

2.  Wash  in  running  water.     Blot  up  the  excess. 

3.  Pass  rapidly  through  absolute  alcohol  to  xylol  and  mount  in  balsam. 
The  bacilli  are  stained  red  and  the  tissues  of  the  section  blue  or  violet. 

B.  Saathoff  s  method. — This  is  a  modification  of  the  preceding  rendering 
the  latter  more  convenient  and  yielding  preparations  which  keep    better. 
Alcohol  may  be  used  to  fix  the  tissues. 

1.  Stain  for  about  4  minutes  in  the  following  solution  which  must   be 
filtered  before  use  : 


Methyl-green, 

Pyronin, 

96  per  cent,  alcohol, 

Glycerin, 

2  per  cent,  aqueous  solution  of  carbolic  acid, 


0'15  gram. 
0-5        „ 
5       grams. 

20 

Q.S.  ad  100  c.c. 


2.  Wash  in  running  water  until  the  green  colour  gives  place  to  a  bluish- 
red.     Blot  up  the  excess  of  water. 

3.  Dehydrate  very  rapidly  in  absolute  alcohol.     Wash  in  xylol.     Mount 
in  balsam. 


CHAPTER  XIV. 

IMMUNITY.1 

THE  PROPERTIES  OF  IMMUNE  SERUMS. 

Introduction. — The  mechanism  of  immunity,  p.  222. 
Section  I. — Prophylactic  and  therapeutic  serums,  p.  223. 
Section  II. — Antitoxins,  p.  224. 
Section  III. — Agglutinins,  p.  225. 

The  mechanism  of  agglutination,  p.  226. 
Section  IV. — Bactericidal  properties,  p.  227. 

The  mechanism  of  bacteriolysis,  p.  228.     Haemolysins,  p.  230.     The  mechanism  of 
haemolysis,  p.  231.     The  fixation  of  the  complement,  p.  232. 
Section  V.— Opsonins,  p.  239. 

IMMUNITY  as  the  word  is  applied  in  bacteriology  denotes  the  faculty 
possessed  by  a  living  animal  of  resisting  an  infection  or  intoxication. 

Immunity  to  a  particular  organism  or  toxin  may  be  natural  or  acquired. 

Natural  immunity  is  a  function  of  the  species  and  only  rarely  of  the  race. 
In  some  cases  it  has  a  relation  to  age  :  thus,  adults  may  be  immune  while 
the  young  of  the  same  species  are  susceptible  to  a  particular  infection  or 
intoxication.  Again  immunity  may  be  absolute  or  relative. 

Acquired  immunity  to  a  specific  disease  may  be  a  natural  condition  resulting 
from  an  attack  of  that  disease ;  for  instance,  a  person  rarely  suffers  from 
more  than  one  attack  of  enteric  fever,  measles  or  anthrax  ;  or  it  may  be  a 
condition  artificially  produced  in  an  individual  in  response  to  the  inoculation 
of  a  virus,  a  toxin,  or  the  serum  of  an  immunized  animal. 

Immunity  artificially  produced  may  be  active  or  passive. 

Active  immunity  is  the  result  of  the  inoculation  of  small  doses  of  vigorous 
cultures  of  living  organisms,  of  cultures  of  living  organisms  attenuated  either 
by  heat  or  by  prolonged  artificial  cultivation,  of  dead  organisms,  or  of  the 
toxins  which  organisms  produce.  An  active  reaction  takes  place  in  the  living 
tissues  in  response  to  the  inoculation  with  the  result  that  the  subject  has 
acquired  certain  new  properties  and  these  will  have  to  be  studied  in  detail. 
Active  immunity  is  only  acquired  slowly  and  then  at  the  cost  of  a  real  and 
occasionally  serious  disease  during  which  the  tissues  may  be  highly  susceptible 
to  further  inoculation  of  the  particular  virus ;  but  on  the  other  hand  the 

1  It  would  obviously  be  beyond  the  scope  of  a  book  such  as  this  to  enter  into  a  detailed 
study  of  immunity  and  the  theories  associated  with  it.  The  present  chapter  is  therefore 
limited  to  such  explanations  as  are  indispensable  to  the  proper  understanding  of  the 
subsequent  chapters  and  to  an  account  of  the  principal  methods  of  demonstrating  the 
properties  of  immune  serums. 


222  THE   MECHANISM   OF  IMMUNITY 

immunity  so  acquired  is  lasting  and  occasionally  absolute.  By  increasing  the 
number  of  successive  vaccinating  inoculations  the  animal  may  in  time  become 
so  highly  immunized  that  even  enormous  doses  of  the  specific  organism  or 
toxin  have  no  visible  effect  upon  it :  this  is  a  special  condition  of  hyper- 
immunization  in  which  the  resistance  of  the  animal  is  raised  to  its  highest 
limits. 

But  if  a  non-immune  subject  be  inoculated  with  the  serum  of  an  immunized 
or  hyper-immunized  animal  instead  of  with  organisms  or  toxins  a  different 
result  ensues.  The  former  is  certainly  rendered  immune  but  in  this  case  it 
is  merely  a  condition  of  passive  immunity.  The  person  or  animal  passively 
immunized  has  taken  no  active  part  in  the  process  of  immunization  but  has 
simply  been  inoculated  with  something  possessing  prophylactic  properties. 
The  period  during  which  such  immunity  lasts,  which  is  always  very  short 
(generally  a  few  days  only),  is  dependent  upon  the  time  during  which  the 
substance  inoculated  remains  in  the  tissues  and  as  soon  as  it  is  eliminated 
the  immunity  has  gone. 

The  mechanism  of  immunity. 

If  a  living  animal  be  immune  against  a  pathogenic  organism,  the  inocula- 
tion of  that  organism  into  the  animal  results  in  an  aggregation  of  leucocytes  at 
the  site  of  inoculation  (chemiotaxis)  which  ingest  and  digest  the  inoculated 
organisms.  This  is  the  phenomenon  described  by  Metchnikoff  as  phago- 
cytosis. 

Phagocytosis  can  be  easily  observed,  for  instance,  with  the  anthrax  bacillus. 
If  a  healthy  guinea-pig  be  inoculated  with  a  trace  of  an  anthrax  culture  the 
tissues  about  the  site  of  inoculation  soon  become  the  seat  of  an  cedematous 
infiltration  (the  oedema  consists  of  a  serous  fluid  containing  free  organisms 
but  very  few  leucocytes)  :  the  bacillus  quickly  generalizes  and  death  rapidly 
supervenes.  On  the  other  hand,  if  a  guinea-pig  previously  vaccinated 
against  anthrax  be  inoculated  it  can  be  shown  that  numbers  of  leucocytes 
very  rapidly  accumulate  at  the  site  of  inoculation  and  in  a  few  hours  have 
ingested,  killed  and  digested  all  the  bacilli,  the  animal  suffering  no  ill-effects 
from  the  inoculation.  A  similar  observation  can  be  made  on  dogs,  animals 
naturally  immune  to  anthrax.  The  inoculation  of  anthrax  bacilli  into  dogs 
is  followed  by  a  small  abscess  in  which  phagocytosis  is  very  active  but  the 
infection  does  not  become  generalized.1 

The  leucocytes  take  up  the  micro-organisms  while  the  latter  are  still  living. 
Experiments  have  been  devised  to  show  that  organisms  ingested  by  leucocytes 
retain  their  vitality  for  a  greater  or  lesser  length  of  time  during  which  they 
can,  in  a  non-immune  animal,  set  up  a  fatal  infection  (Metchnikoff). 

On  the  other  hand,  in  some  cases,  notably  in  the  case  of  the  cholera  vibrio, 
it  has  been  observed  that  if  the  vibrio  be  inoculated  into  the  peritoneal  cavity 
of  an  immunized  guinea-pig  it  is  killed  not  after  ingestion  by  the  leucocytes — 
which  are  present  in  very  small  numbers  in  the  exudate— but  in  the  exudate 
itself  :  this  constitutes  Pfeiffer's  phenomenon  (vide  infra).  Such  a  phenomenon 
might  be  quoted  as  an  objection  to  the  theory  of  phagocytosis  but  more 
extended  observation  shows  bactericidal  action  of  this  nature  by  the  body 
fluids  to  be  exceptional :  it  may  be  described  as  a  make-shift  in  the  defence 
of  the  individual  and  only  occurs  when  the  leucocytes  have  undergone  changes 
which  prevent  them  coming  in  contact  with  the  organisms  themselves  and 
is  moreover  only  seen  in  the  case  of  a  few  very  delicate  organisms. 

1  Micro-organisms  have  their  own  means  of  defence  in  their  fight  with  the  leucocytes  : 
they  secrete  soluble  substances,  agressins,  which  act  on  the  white  cells  of  the  blood  and 
prevent  them  ingesting  and  destroying  the  infecting  agents.  In  conditions  of  immunity 
the  leucocytes  triumph  over  these  agressins  and  thus  fulfil  their  function  of  defence. 


PROPHYLACTIC  SERUMS  223 


According  to  Metchnikoff  the  bactericidal  substances  in  the  serum  are  derived 
from  the  leucocytes  :  some  (immune  bodies,  amboceptors,  or  sensibilisatrices)  are 
elaborated  in  the  leucocytes  and  excreted  into  the  plasma  as  they  are  formed,  whence 
they  pass  into  the  different  tissues  of  the  animal ;  the  others  (complement,  cytase 
or  alexin)  are  also  of  leucocytic  origin  but  are  only  set  free  on  the  death  and  dis- 
integration of  the  leucocytes.  Petterson  and  Schneider  consider  that  there  are 
yet  other  substances  in  the  leucocytes  capable  of  destroying  micro-organisms  (endo- 
lysins,  leukins). 

In  the  majority  of  cases  the  bactericidal  substances  of  the  serum  of  im- 
munized animals  intervene  to  prepare  the  micro-organisms  for  the  action  of 
the  leucocytes  and  facilitate  their  ingestion  and  destruction  (vide  opsonins). 

In  immunized  animals  therefore  over  and  above  the  phagocytic  reaction 
there  exist  in  the  fluid  part  of  the  blood  (serum)  certain  substances  of  great 
importance  which  play  a  prominent  part  in  the  phenomena  of  immunity. 
The  properties  of  these  immune  serums  will  be  now  studied  a  little  more  fully. 

The  serums  of  immunized  animals  may  exhibit  one  or  more  or  all  of  the 
following  properties  each  quite  independently  of  the  other  and  in  different 
degrees. 

1.  Prophylactic  and  therapeutic  properties. 

2.  Antitoxic  properties. 

3.  Agglutinating  properties. 

4.  Bactericidal  properties. 

5.  The  property  of  preparing  micro-organisms  for  ingestion  by  the  leuco- 
cytes.    This  property  which  is  due  to  the  presence  of  special   substances, 
opsonins,  would  appear  to  be  connected  with  the  bactericidal  properties. 


SECTION  I.— PROPHYLACTIC  AND   THERAPEUTIC  SERUMS. 

It  has  already  been  pointed  out  that  the  serum  of  an  animal  vaccinated 
against  a  micro-organism  if  inoculated  into  a  fresh  animal  confers  on  the 
latter  an  immunity  of  short  duration. 

This  passive  immunization  is  absolutely  specific  and  is  only  exhibited 
towards  the  species  of  organism  with  which  the  first  animal  was  vaccinated. 

The  serum  of  an  animal  vaccinated  with  toxin  if  inoculated  into  a  fresh 
animal  confers  on  the  latter  an  immunity  against  the  same  toxin  and  also 
against  the  micro-organism  which  elaborated  the  toxin. 

Example. — If  a  normal  guinea-pig  be  inoculated  with  antidiphtheria  serum  it  is 
protected  against  the  inoculation  of  diphtheria  •  toxin  and  also  against  inoculation 
with  the  diphtheria  bacillus. 

On  the  other  hand,  if  an  animal  be  vaccinated  with  micro-organisms  its 
serum  has  no  protective  action  against  the  toxin  of  the  organism  though 
it  protects  against  the  organism  itself. 

Example. — The  serum  of  an  animal  vaccinated  with  the  cholera  vibrio  (vide 
Cholera)  will  protect  a  normal  animal  against  an  inoculation  of  the  vibrio.  A 
trace  of  the  serum,  for  instance,  inoculated  into  a  normal  guinea-pig  will  vaccinate 
the  latter  against  choleraic  peritonitis.  On  the  other  hand  the  serum  affords  no 
protection  against  an  inoculation  of  the  toxin  and  is  totally  ineffective  in  intestinal 
cholera  which  is  an  intoxication  (Metchnikoff). 

In  all  of  the  foregoing  cases  the  serum  acts  as  a  prophylactic  ;  that  is  to 
say,  it  immunizes  the  animal  to  which  it  is  administered  provided  it  be  inocu- 
lated before  or  at  the  same  time  as  the  organisms  or  toxin. 

Some  serums  exhibit  therapeutic  as  well  as  prophylactic  properties.  If 
inoculated  after  the  infection,  even  though  the  first  symptoms  of  infection 
have  appeared,  they  abort  the  disease  and  lead  to  recovery.  The  curative 


224  ANTITOXINS 

properties  of  a  serum  do  not  always  run  parallel  with  its  prophylactic  pro- 
perties. To  quote  a  classical  instance :  antidiphtheria  serum  is  both 
prophylactic  and  curative,  but  antitetanus  serum  while  exhibiting  very 
marked  prophylactic  properties  has  no  curative  properties.  These  pro- 
perties of  immune  serums  will  be  referred  to  again  in  more  detail,  each  serum 
being  dealt  with  in  connexion  with  its  corresponding  organism. 

SECTION  II.— ANTITOXINS. 

If  an  animal  be  inoculated  with  progressively  increasing  doses  of  a  micro- 
organic  toxin  it  will  ultimately  become  immunized  against  this  toxin,  and 
will  be  able  to  tolerate  without  suffering  any  inconvenience  doses  infinitely 
greater  than  those  which  if  given  in  the  first  instance  would  have  proved 
fatal  (Behring  and  Kitasato). 

To  this  general  rule  there  are  however  a  few  exceptions  and  these  have  been 
described  by  Richet  as  cases  of  anaphylaxis. 

Richet  showed  that  if  a  dog  were  inoculated  with  a  small  dose  of  actino-con- 
gestine  (the  poison  in  the  tentacles  of  sea  anemones)  it  exhibited  no  ill-effects  ; 
but  if  10-20  days  after  the  first  inoculation  it  were  re- inoculated  with  the  same  or 
even  with  a  smaller  dose  than  that  which  before  proved  harmless  the  animal 
quickly  died.  This  result  cannot  be  explained  on  the  theory  of  an  accumulation  of 
toxin  because  the  whole  quantity  given  in  the  two  doses  is  very  much  less  than  that 
which  would  be  required  to  produce  a  fatal  result  if  given  in  the  first  instance,  and 
further  if  the  second  inoculation  be  given  from  1—6  days  after  the  first,  the  animal 
does  not  die  :  the  phenomena  of  anaphylaxis  do  not  appear  until  about  the  tenth 
day.  The  serum  of  an  anaphylactic  dog  inoculated  into  a  normal  dog  produces  a 
condition  of  hypersensibility  immediately  after  inoculation,  and  hence  the  serum 
of  anaphylactic  animals  contains  the  substance — whatever  its  nature — causing  the 
phenomena  of  anaphylaxis  (Richet). 

Other  instances  of  anaphylaxis  may  be  quoted.  If  an  animal  be  inoculated  once 
with  the  serum  of  another  species  it  is  only  rarely  and  then  inconstantly  that  any 
untoward  symptoms  develop,  but  if  successive  re-inoculations  be  made  the  result 
is  quite  different,  the  reaction  on  the  part  of  the  inoculated  animal  being  then  very 
violent  and  likely  to  terminate  fatally  (Arthus).  This  phenomenon  is  seen  for 
example  when  rabbits  or,  better,  guinea-pigs,  are  repeatedly  inoculated  with  horse 
serum.  According  to  von  Pirquet  and  Schrick  the  grave  symptoms  occasionally 
observed  in  the  human  subject  after  injections  of  antidiphtheria  serum  are  of 
an  anaphylactic  nature. 

Anaphylaxis  in  connexion  with  tuberculosis  has  also  been  the  subject  of  experi- 
mental observation.  The  reaction  to  tuberculin  is  an  anaphylactic  phenomenon  : 
the  inoculation  of  a  trace  of  tuberculin  into  man  or  an  animal  affected  with  tuber- 
culosis sets  up  a  severe  reaction  (vide  Tuberculosis)  and  numerous  methods  of 
diagnosis  are  based  on  this  reaction. 

Still  further  examples  of  anaphylaxis  could  be  given  but  it  must  suffice  here  to 
have  drawn  attention  to  the  existence  of  this  phenomenon.  To  investigate  the 
mechanism  of  anaphylaxis  and  to  discuss  the  theories  which  have  been  advanced  in 
explanation  of  it  would  be  altogether  beyond  the  scope  of  the  present  work. 

The  serum  of  animals  which  have  survived  the  inoculation  of  repeated  and 
increasing  doses  of  toxin  has  acquired  antitoxic  properties. 

Antitoxin,  like  toxin,  has  its  nature  altered  by  being  heated,  is  precipitated 
by  alcohol,  and  is  carried  down  by  precipitates  formed  in  the  liquid  in  which 
it  is  in  solution.  In  suitable  quantities  it  saturates  toxin  both  in  the  tissues 
and  in  vitro.  In  mixtures  in  vitro  toxin  is  not  destroyed  by  antitoxin  but  is 
simply  disguised  ;  the  toxin-antitoxin  mixture  is  nevertheless  harmless  to 
animals,  though  under  certain  conditions  the  poisonous  nature  of  the  toxin  may 
be  made  to  reappear ;  thus,  if  a  neutral  mixture  of  snake  venom  and  anti- 
venomous  serum  be  heated  to  70°  C.  the  antitoxin  is  destroyed  but  not  the 
toxin  so  that  the  mixture  is  now  no  longer  harmless. 


AGGLUTININS  225 


Antitoxic  serums  are  strictly  specific.  Under  the  head  of  each  of  the 
pathogenic  micro-organisms  the  antitoxic  properties  of  the  corresponding 
serum  will  be  considered  in  detail. 


SECTION  III.— AGGLUTININS. 

Durham  and  Gruber  when  studying  antityphoid  serum  demonstrated  a 
very  remarkable  property  of  the  serum.  If  a  small  quantity  of  serum  from 
a  typhoid-immunized  animal  be  added  to  a  broth  culture  of  the  typhoid 
bacillus  the  bacilli  distributed  through  the  medium  lose  their  motility,  collect 
together  and  become  agglutinated  into  masses,  retaining  however  their 
vitality.  This  phenomenon  is  known  as  agglutination  and  the  serum  is  said 
to  possess  agglutinating  properties. 

Previously  to  Durham  and  Gruber's  experiments,  Bordet  had  demonstrated 
a  similar  action  of  anticholera  serum  on  cholera  vibrios,  and  it  has  since 
been  shown  that  in  the  majority  of  cases  the  serum  of  an  animal  immunized 
against  a  micro-organism  agglutinates  the  organism  used  for  immunization 
(cholera,  dysentery,  tuberculosis,  mediterranean  fever,  plague,  glanders,  etc.). 

The  property  of  agglutination  however  is  not  limited  to  the  serum  of 
immunized  animals.  [A.  S.  Griinbaum  showed  that]  it  appears  quite  early, 
before  a  state  of  immunity  has  been  created,  as  soon  as  the  tissues  have 
been  invaded  by  a  pathogenic  organism.  The  reaction  of  agglutination  is  a 
reaction  of  infection.  It  remains,  moreover,  for  a  long  time  after  recovery 
has  taken  place,  being  found  as  has  already  been  stated  in  a  marked  degree 
in  the  serum  of  immunized  individuals. 

The  agglutination  reaction  is  specific  :  the  serum  of  an  enteric  patient 
agglutinates  the  typhoid  bacillus  and  (with  certain  reservations)  the  typhoid 
bacillus  only.  The  serum  of  cholera  patients  similarly  agglutinates  only  the 
cholera  vibrio. 

[A.  S.  Griinbaum  and  shortly  afterwards]  Widal  showed  that  practical  use 
can  be  made  of  these  facts  in  the  diagnosis  of  infective  diseases  and  to  [the 
former]  is  due  the  method  of  serum  diagnosis.  Take,  for  example,  the  case 
of  a  person  thought  to  be  suffering  from  enteric  fever  :  it  is  only  necessary 
to  mix  a  few  drops  of  his  serum  with  a  culture  of  the  typhoid  bacillus  : 
then  if  the  patient  be  suffering  from  enteric  fever  the  bacilli  will  be 
agglutinated  ;  on  the  other  hand,  if  he  be  suffering  from  some  disease  other 
than  enteric  fever  the  bacilli  will  remain  separate  and  motile. 

Conversely,  suppose  it  is  required  to  determine  whether  a  bacillus  is  the 
typhoid  bacillus  or  not :  in  this  case  it  is  sufficient  to  prepare  a  culture  of 
the  unknown  bacillus  and  to  mix  it  [in  due  proportion]  with  a  typhoid - 
agglutinating  serum  :  if  agglutination  take  place  the  bacillus  may  without 
hesitation  be  affirmed  to  be  the  typhoid  bacillus. 

To  obtain  reliable  results,  there  are  certain  precautions  which  must  be 
strictly  observed  in  carrying  out  the  reaction.  To  exemplify  :  most  normal 
serums — and  especially  human  serums — when  used  in  large  quantities 
agglutinate  a  considerable  number  of  organisms  :  if  a  mixture  of  serum  and 
organisms  be  made  without  knowing  the  proportions  in  which  they  are  mixed 
agglutination  might  be  obtained  apart  from  any  specific  relation  of  the 
ingredients  to  each  other.  The  following  rules  should  therefore  always  be 
followed  : 

(i)  The  serum  under  investigation  must  be  diluted  [Grunbaum]  and  the 
dilution  carried  to  such  a  degree  that  the  minimal  dose  of  serum  required  for 
lutination  is  determined.  For  purposes  of  comparison  the  minimum 

p 


226  MECHANISM  OF  AGGLUTINATION 

quantity  of  normal  serum  (human  or  animal)  required  to  produce  agglutina- 
tion must  also  be  determined. 

For  example,  it  can  be  shown  that  while  normal  serum  frequently  aggluti- 
nates the  typhoid  bacillus  in  a  dilution  of  1  in  10  a  typhoid  serum  will 
agglutinate  it  in  dilutions  of  1  in  200,  1  in  500,  and  even  in  1  in  5,000. 

(ii)  It  becomes  even  more  imperative  to  dilute  the  serum  when  it  is 
recognized  that  a  specific  serum  will  agglutinate  not  only  its  corresponding 
organism  but  also,  not  infrequently,  closely  related  species,  provided  that 
the  quantity  of  serum  used  be  sufficient  [Griinbaum].  It  is  obvious  therefore 
that  unless  a  serum  be  adequately  diluted  its  specific  characters  will  escape 
recognition. 

Take  an  example  :  a  patient  is  suffering  from  a  para-typhoid  infection. 
His  serum  agglutinates  both  the  typhoid  and  the  para-typhoid  bacillus  in 
dilutions  of  1  in  20  and  1  in  50  :  so  far  there  is  nothing  specific  about  the 
serum.  Dilute  the  serum  further,  say  to  1  in  100,  1  in  200,  and  1  in  500.  In 
these  higher  dilutions  it  has  entirely  lost  all  its  agglutinating  property  for 
the  typhoid  bacillus  but  still  agglutinates  the  para-typhoid  bacillus.  In 
this  case  the  specific  nature  of  the  agglutination  is  determined  by  the  titre 
of  agglutination  and  not  by  the  mere  fact  of  agglutination  itself. 

(iii)  It  is  also  of  the  highest  importance  in  studying  the  phenomena  of 
agglutination  that  only  homogeneous  emulsions  or  cultures  be  used  in  which 
the  organisms  are  as  far  as  possible  lying  separately,  for  if  they  be  clumped 
or  massed  together  the  results  of  the  experiments  will  obviously  be  misleading. 
This  spontaneous  clumping  is  a  source  of  great  difficulty  when  working  at 
agglutination  with  organisms  which  naturally  grow  in  clumps.  The  difficulty 
may  be  overcome  either  by  using  very  young  cultures  in  broth  (typhoid 
bacillus)  or  by  having  resort  to  one  or  other  of  the  various  methods  which 
have  been  devised  for  obtaining  homogeneous  cultures  (of  the  tubercle 
bacillus,  etc.). 

(iv)  Finally,  in  performing  agglutination  tests  with  serums  care  must  be 
taken  to  add  the  serum  to  the  culture  and  never  to  add  the  culture  to  the 
serum.  It  can  be  easily  understood  that  in  the  latter  case  the  first  drops  of 
culture  would  be  mixed  with  an  undiluted  serum  and  that  agglutinated 
masses  of  organisms  might  form  even  though  there  were  no  specific  relation- 
ship between  the  organism  and  the  serum. 

The  technique  of  serum  diagnosis  will  be  described  in  detail  in  the  chapter 
on  the  typhoid  bacillus,  and  under  the  head  of  each  micro-organism  data 
with  regard  to  agglutination  will  be  given. 

The  mechanism  of  agglutination. 

It  would  appear  that  the  phenomena  of  agglutination  are  not  dependent 
upon  any  vital  activity  of  the  organisms  since  they  can  be  observed  with 
dead  cultures. 

The  substances  in  serums  producing  agglutination  are  known  as  agglutinins. 
Agglutinins  are  distinguishable  from  bactericidal  substances  in  that  unlike 
the  latter  they  withstand  heating  at  55°  C.  for  half  an  hour  and  are  only 
destroyed  at  about  60°  C.  in  serum  and  70°  or  80°  C.  in  milk.  They  are  pre- 
cipitated by  alcohol  and  do  not  pass  through  a  Chamberland  or  Berkefeld 
bougie.  But  since  they  can  be  demonstrated  in  the  milk,  urine,  etc.,  of 
infected  or  immunized  animals  it  would  appear  that  they  can  pass  through 
certain  living  animal  membranes. 

The  phenomena  of  agglutination  may  be  explained  on  the  assumption 
that  the  agglutinin  acts  on  some  agglutinatible  substance  present  in  the  bodies 
of  the  organisms  agglutinated.  Organisms  which  have  been  separated  from 


PRECIPITINS  227 


the  culture  medium  by  filtration,  washed  and  suspended  in  normal  saline 
solution  still  retain  the  property  of  being  agglutinated  by  a  specific  serum. 
But,  as  Kraus  and  Ch.  Nicolle  have  shown,  if  a  culture  be  filtered  through 
porcelain  a  flocculent  precipitate,  similar  to  masses  of  agglutinated  micro- 
organisms, forms  on  the  addition  of  a  specific  serum  to  the  filtrate.  It  is 
obvious  therefore  that  the  agglutinatible  substance  is  also  present  in  the 
culture  fluid  ;  it  may  be  that  as  the  organisms  grow  old  the  agglutinatible 
substance  passes  into  the  culture  fluid.  The  name  precipitins  has  been 
suggested  for  the  substances  in  serum  which  cause  the  precipitate  in  filtered 
cultures  :  there  is  evidence  that  precipitins  and  agglutinins  are  identical 
bodies. 

Finally,  certain  chemical  substances  have  the  property  of  agglutinating 
micro-organisms  (Malvoz)  but  their  action  is  in  no  way  specific  and  the 
same  substance  will  agglutinate  different  micro-organisms  (Beco).  A  mixture 
of  equal  parts  of  commercial  formalin,  alcohol,  hydrogen  peroxide,  a  1  in 
1,000  solution  of  chrysoidin,  vesuvin,  safranin,  or  perchloride  of  mercury,  etc., 
agglutinates  the  typhoid  bacillus  as  well  as  various  other  organisms. 


SECTION  IV.— BACTERICIDAL  PROPERTIES. 

The  fact  that  the  serum  of  immunized  animals  has  the  power  of  destroying 
bacteria  was  brought  to  light  by  one  of  Pfeiffer's  experiments  which  has 
since  become  classical. 

Pfeiffer's  experiment. — If  a  normal  guinea-pig  be  inoculated  in  the  peri- 
toneal cavity  with  a  broth  culture  of  the  cholera  vibrio  the  animal  rapidly 
succumbs  from  peritonitis,  and  if  the  peritoneal  exudate  be  examined  micro- 
scopically in  a  hanging-drop  preparation  it  is  found  to  contain  very  large 
numbers  of  free  motile  vibrios,  exactly  similar  to  those  inoculated. 

Let  the  same  experiment  be  done  on  a  guinea-pig  which  has  been  immunized 
against  the  cholera  vibrio ;  the  animal  survives  the  inoculation  and  an 
examination  of  the  peritoneal  fluid  reveals  an  entirely  different  condition. 

In  a  drop  of  the  fluid  removed  10-30  minutes  after  the  inoculation  it  will 
be  found  that  not  only  have  the  vibrios  not  multiplied  but  they  have  also 
lost  their  motility,  and  instead  of  finding  numerous  elongated  comma-shaped 
organisms  as  in  the  former  case,  the  fluid  is  seen  to  contain  small  granules 
of  no  definite  shape,  which  soon  disappear  altogether  being  destroyed  in  the 
fluid  in  which  they  are  suspended. 

This  granular  metamorphosis  followed  by  complete  destruction  of  the 
vibrio  may  also  be  demonstrated  in  vitro  (Bordet,  Metchnikoff). 

Bordet's  experiment. — Break  up  a  small  quantity  of  an  agar  culture  of  the 
cholera  vibrio  in  a  little  sterile  broth  :  examine  the  emulsion  under  a  micro- 
scope to  see  that  there  are  no  granular  forms  and  that  the  vibrios  are  quite 
motile  :  add  to  the  emulsion  ^V~TVth  °f  its  volume  of  the  serum  of  an  im- 
munized guinea-pig.  On  examining  the  mixture  a  few  minutes  after  the 
addition  of  the  serum,  the  vibrios  will  be  seen  to  have  lost  their  motility  and 
to  have  become  agglutinated  and  converted  into  granular  dots :  the  re- 
action is  however  not  at  its  maximum  until  the  mixture  has  been  kept  at 
37°  C.  for  1  or  2  hours. 

From  these  two  experiments  it  may  be  concluded  that  the  serum  of 
immunized  guinea-pigs,  apart  from  the  intervention  of  any  cellular  element, 
contains  bactericidal  and  bacteriolytic  substances  capable  of  destroying  the 
cholera  vibrio. 

These  substances  are  specific  so  that  the  serum  is  only  bactericidal  for  the 


228  BACTERIOLYSINS 

organism  with  which  the  animal  has  been  immunized.  The  serum  of  animals 
immunized  with  the  typhoid  bacillus  for  instance  is  bactericidal  only  for  the 
typhoid  bacillus  and  is  totally  devoid  of  action  on  the  cholera  vibrio,  and, 
vice  versa,  an  anticholera  serum  is  not  bactericidal  for  the  typhoid  bacillus. 

The  bactericidal  action  of  immunized  serums  is  rapid  and  at  its  maximum 
at  37°  C.,  feeble  at  the  ordinary  temperature  of  the  laboratory  and  altogether 
paralyzed  at  0°  C. 

The  analysis  of  the  phenomena  of  bacteriolysis  may  now  be  pushed  a  step 
further  and  an  attempt  made  to  investigate  the  mechanism  by  which  bacterio- 
lysis occurs. 

Mechanism  of  bacteriolysis. 

Suppose  the  serum  of  a  guinea-pig  immunized  with  the  cholera  vibrio  be 
heated  to  55°  C.  and  then  mixed  with  a  culture  of  the  vibrio.  Bacteriolysis 
no  longer  takes  place,  though  the  agglutinating  properties  of  the  serum 
remain  unaffected  (Bordet). 

The  heated  serum,  however,  has  not  altogether  lost  its  bactericidal  pro- 
perties; for,  if  to  the  mixture  of  vibrios  and  heated  serum  a  small  quantity 
of  serum  from  a  normal  animal  be  added,  bacteriolysis  occurs  at  once — the 
heated  serum  is  re-activated. 

It  may  therefore  be  concluded  that  the  serum  of  the  immunized  animal 
contains  two  substances  : 

(i)  One  of  which  is  not  destroyed  by  being  heated  at  55°  C.  or,  in  other  words 
is  thermostable  at  55°  C.  and  which  is  only  present  in  the  serum  of  immunized 
animals. 

(ii)  The  other  of  which  is  destroyed  by  heating  to  55°  C.  or,  in  other  words, 
is  thermolabile  at  55°  C.  and  which  is  present  also  in  the  serum  of  normal 
animals. 

These  two  substances  when  present  together  cause  bacteriolysis  but  either 
the  one  or  the  other  acting  alone  has  no  action  on  the  vibrio.  Let  us  con- 
sider now  the  part  which  each  of  these  substances  plays. 

A.  Immune  body  (Sensibilisatrice). — The  thermostable  substance  is,  as  has 
been  said,  present  only  in  the  serum  of  immunized  animals  and  is  a  product 
of  immunization  [hence  the  term  immune  body  generally  used  in  England]. 
And,  further,  it  is  specific  and  acts  only  on  the  organism  which  was  used  for 
immunization. 

Experiment. — Treat  an  emulsion  of  cholera  vibrios  with  anticholera  serum  heated 
to  55°  C.  and  then  add  a  little  normal  rabbit  serum.  The  vibrios  will  be  bac- 
teriolyzed. 

Repeat  the  experiment  using  instead  of  cholera  vibrios,  typhoid  bacilli.  Treat 
the  typhoid  bacilli  with  heated  (55°  C.)  anticholera  serum  and  then  add  the  normal 
rabbit  serum.  No  bacteriolysis  takes  place. 

The  immune  body  in  contact  with  its  corresponding  micro-organism  is 
fixed  by  the  organism  in  the  same  way  that  a  mordant  acts  on  a  fabric. 

Experiment. — Leave  a  mixture  of  cholera  vibrios  and  heated  anticholera  serum 
for  half  an  hour  at  37°  C.  Centrifuge  to  separate  the  vibrios,  wash  the  latter  with 
normal  saline  solution  and  then  add  a  little  fresh  normal  rabbit  serum  to  the  vibrios. 
Bacteriolysis  takes  place. 

This  experiment  justifies  the  view  held  by  Bordet  that  the  immune  body 
sensitizes  the  organisms  to  the  action  of  the  thermolabile  substance  present 
in  normal  serum  just  as  a  mordant  sensitizes  a  fabric  to  the  action  of  a  dye. 
Hence  the  use  in  France  of  the  word  Sensibilisatrice  to  denote  the  immune 
body.  The  immune  body  is  only  destroyed  by  heating  it  at  65°-70°  C. 

Sensitized  micro-organisms,  that  is  to  say,  organisms  which  have  been 


MECHANISM  OF  BACTERIOLYSIS 


229 


treated  with  their  specific  immune  body,  continue  to  grow  in  the  ordinary 
way  and  have  lost  none  of  their  pathogenic  properties,  but  they  differ  from 
non-sensitized  organisms  not  only  in  their  susceptibility  to  the  action  of  the 
thermolabile  substance  or  complement-  (vide  infra)  present  in  all  normal 
serums,  but  also  in  that  they  are  more  easily  ingested  and  destroyed  by 
leucocytes. 

The  immune  body  is  known  by  different  names,  substance  sensibilisatrice 
(Bordet),  fixateur,   amboceptor   (Ehrlich).     Occasionally   it   is   described   as 


i  c 

FIG.  159. — Ehrlich's  diagram  to  explain  the  interaction  between  the  immune 
body  and  complement. 

A,  micro-organism  or  antigen ;  /,  immune  body,  sensibilisatrice,  amboceptor, 
flxateur  or  antibody ;  C,  complement,  alexin  or  cytase. 

the  antibody  because  it  is  antagonistic  to  the  substances  inoculated  into 
animals  for  the  purpose  of  immunizing  them.  These  latter  substances  are 
therefore  called  antigens.  Thus,  in  immunizing  animals  against  the  typhoid 
bacillus  the  antigen  is  the  typhoid  bacillus  and  the  antibody  the  new  product 
appearing  in  the  serum  in  response  to  the  inoculation  of  the  antigen  and 
which  has  the  property  of  attaching  itself  to  the  typhoid  bacillus  and  so  of 
rendering  the  bacillus  susceptible  to  bacteriolysis. 

B.  Complement. — The  immune  body  prepares  organisms  for  the  action  of 
the  substance  contained  in  the  serum  of  normal  animals.  This  latter  sub- 
stance in  conjunction  with  the  immune  body  produces  bacteriolysis,  hence 
the  name  complement  by  which  it  is  generally  described  (Ehrlich).  By  some 
authors,  however,  it  is  occasionally  referred  to  as  alexin  (Bordet)  or  cytase 
(Metchnikoff  and  Buchner). 

The  complement  is  not  a  product  of  immunization  and  is  not  specific  but 
is  present  in  all  normal  serums  and  is  fixed  indifferently  by  all  organisms 
through  their  specific  immune  body. 

When  complement  is  mixed  with  micro-organisms  it  is  only  taken  up  by 
them  if  they  have  been  sensitized.  Complement  has  no  affinity  for  non- 


230  ILEMOLYSINS 

sensitized  organisms  and  remains  in  the  serum.     Its  action  is  like  that  of  a 
dye  which  only  dyes  a  fabric  that  has  been  treated  with  a  mordant. 

To  sum  up  :  in  the  serum  of  immunized  animals  a  specific  substance, 
the  immune  body,  is  present  which  unites  on  the  one  hand  with  its 
corresponding  micro-organism  and  on  the  other  hand  with  a  substance, 
alexin  or  complement,  pre-existing  in  the  serum  of  all  normal  animals. 
Upon  this  interaction  of  two  bodies,  in  which  the  immune  body  plays  a 
part  similar  to  that  of  a  mordant  in  dyeing,  depends  the  destruction  of 
the  micro-organisms  in  Pfeiffer's  experiment. 

The  interaction  of  complement  with  the  micro-organism  through  the  immune 
body  is  diagrammatically  represented  in  the  figure  (fig.  159). 

Note. — It  is  important  to  observe  that  the  phenomena  just  studied  are  not 
seen  in  the  case  of  all  micro-organisms.  The  combined  action  of  the  immune 
body  and  complement  only  leads  to  destruction  in  the  case  of  very  delicate 
organisms,  e.g.  the  cholera  vibrio  and  the  typhoid  bacillus. 

In  the  majority  of  cases  the  pathogenic  micro-organisms  are  much  more 
resistant  to  the  bactericidal  action  of  the  immune  serums  and  no  bactericidal 
action  can  be  seen  ;  but  though  not  visible,  combination  of  the  immune  body 
with  the  complement  nevertheless  takes  place,  and  the  organisms  are  rendered 
more  easy  of  destruction  by  the  leucocytes  (vide  opsonins). 

In  studying  the  phenomena  of  complement  fixation  (vide  infra)  it  will  be 
shown  how  the  action  of  the  immune  body  on  the  more  resistant  organisms 
may  be  demonstrated. 

Haemolysins. 

Haemolysis  means  the  destruction  of  the  red  cells  of  the  blood  with  diffusion 
of  the  haemoglobin  into  the  medium  in  which  they  are  suspended. 

If  a  quantity  of  red  cells  be  suspended  in  an  hypotonic  solution,  distilled 
water  for  example,  they  undergo  haemolysis.  On  the  other  hand,  in  an 
isotonic  fluid,  normal  saline  solution  for  instance,  the  red  cells  may  remain 
intact  for  a  very  long  time.  Similarly  in  the  serum  of  the  majority  of  normal 
animals  the  red  cells  undergo  no  alteration.  To  this  general  rule,  however, 
there  are  a  few  exceptions ;  dogs'  serum,  for  instance,  hsemolyzes  guinea-pig 
red  cells  ;  eel  serum  hsemolyzes  all  mammalian  red  cells,  and  so  on. 

The  inoculation  of  large  doses  of  red  cells  of  one  species  of  animal  into  the 
peritoneal  cavity  of  another  species  produces  a  toxic  effect  and  may  kill  the 
animal  inoculated. 

On  the  other  hand,  if  small  quantities  be  inoculated  on  several  successive 
occasions  there  is  a  minimal  reaction  and  death  does  not  take  place.  In 
the  latter  case  the  serum  of  the  inoculated  animal  is  capable  of  destroying 
the  red  cells  of  the  animal  species  used  for  inoculation  in  vitro  and  is  there- 
fore said  to  exhibit  haemolytic  properties  (Bordet). 

For  example,  if  a  guinea-pig  be  inoculated  with  rabbit  red  cells,  the  guinea- 
pig's  serum  will  become  haemolytic  for  rabbit  red  cells. 

Preparation  of  an  haemolytic  serum. — Under  no  conditions  must  the  whole 
blood  be  inoculated  but  only  the  washed  red  cells. 

1.  Collect  some  blood  under  aseptic  precautions,  and  after  defibrinating  it 
(p.  36)  centrifuge.  Thus,  into  each  tube  of  the  centrifuge  pour  equal  parts 
of  defibrinated  blood  and  sterile  normal  saline  solution,  centrifuge,  and  when 
the  red  cells  are  all  precipitated  at  the  bottom  of  the  tube  pipette  off  the 
clear  supernatant  liquid  with  a  bulb  pipette.  Fill  up  the  tube  with  fresh 
saline  solution,  stir  up  the  deposit  and  centrifuge  again.  Repeat  the  opera- 
tion three  times. 


MECHANISM  OF  HAEMOLYSIS  231 

After  centrifuging  for  the  third  time  dilute  the  red  cells  with  sufficient 
sterile  normal  saline  solution  to  bring  the  total  volume  up  to  the  volume  of 
blood  originally  used. 

It  is  perhaps  unnecessary  to  say  that  these  operations  should  be  carried  out 
under  aseptic  conditions. 

2.  Inoculate  the  animal  (a  guinea-pig  if  using  rabbit  cells,  a  rabbit  for 
sheep  cells,  etc.)  sub-cutaneously,  or  better  into  the  peritoneal  cavity,  on 
five  occasions  at  intervals  of  1  week  with  5-8  c.c.  of  a  suspension  of  red  cells 
prepared  as  described  above.  Experience  has  shown  that  this  amount  does 
not  produce  any  toxic  symptoms.  The  serum  of  the  animal  is  best  collected 
about  1  week  after  the  last  immunizing  inoculation. 

Mechanism  of  haemolysis. 

The  phenomena  of  haemolysis  are  a  counterpart  of  those  of  bacteriolysis. 

The  haemolytic  serum  is  specific  and  only  haemolyzes  red  cells  of  the  animal 
species  used  for  inoculating  the  animal  from  which  the  serum  has  been 
drawn. 

It  contains  two  substances  : 

1.  A  specific  thermostable  immune  body. 

2.  A  complement,   non-specific,  present  in  all  normal  serums  and  only 
becoming  attached  to  the  red  cells  through  the  immune  body.     The  pro- 
perties of  haemolytic  serums  can  be  demonstrated  by  means  of  the  following 
experiments. 

Experimental  illustrations. — Use  the  serum  of  a  guinea-pig  inoculated  with 
rabbit  red  cells  as  an  haemolytic  serum  and  prepare  an  emulsion  of  red  cells 
by  mixing  O'l  c.c.  of  washed  rabbit  red  cells  with  2  c.c.  of  normal  saline 
solution. 

(i)  Mix  the  emulsion  of  red  cells  with  O'l  c.c.  of  the  haemolytic  serum  and 
incubate  at  37°  C.  for  1  hour.  On  taking  the  tube  out  of  the  incubator  it 
will  be  seen  by  simply  looking  at  the  tube  that  haemolysis  is  complete  :  the 
haemoglobin  has  been  discharged  from  the  red  cells  and  imparts  an  uniform 
colour  to  the  solution. 

A  control  tube  in  which  a  little  normal  guinea-pig  serum  has  been  added 
to  an  emulsion  of  red  cells  shows  no  haemolysis ;  the  fluid  contents  are  clear. 

(ii)  To  an  emulsion  of  red  cells,  add  Ol  c.c.  of  haemolytic  serum  previously 
heated  to  55°  C.  for  half  an  hour  and  incubate  at  37°  C. 

The  red  cells  are  not  haemolyzed  but  simply  agglutinated  at  the  bottom 
of  the  tube.  In  this  case  haemolysis  has  failed  because  the  complement  was 
destroyed  by  heating  the  serum  to  55°  C. 

(iii)  To  the  mixture  used  in  the  preceding  experiment  and  which  is  quite 
clear  add  O'l  c.c.  of  normal  guinea-pig  serum  (complement)  and  incubate 
again.  The  red  cells  will  now  undergo  haemolysis. 

(iv)  Kepeat  experiment  (ii)  and  after  showing  that  under  the  conditions  of 
the  experiment  no  haemolysis  occurs,  centrifuge  the  mixture  and  pipette  off 
the  serum  from  the  red  cells. 

(a)  The  centrifuged  serum  has  been  deprived  of  its  immune  body  (which 
has  combined  with  the  red  cells)  and  any  attempt  to  re-activate  it  by  the 
addition  of  complement  (normal  guinea-pig  serum)  fails  ;  it  is  no  longer 
able  to  haemolyze  fresh  rabbit  cells  if  these  be  added  to  it. 

(6)  The  red  cells  separated  from  the  serum  by  centrifuging  have  combined 
with  the  immune  body  ;  so  that  even  after  being  repeatedly  washed  with 
normal  saline  solution  and  centrifuged,  they  are  rapidly  haemolyzed  on  the 
addition  of  O'l  c.c.  of  normal  guinea-pig  serum  (complement)  if  a  mixture  of 
the  two  be  put  in  the  incubator  at  37°  C. 


232  MECHANISM  OF  HAEMOLYSIS 

(v)  If  the  foregoing  experiment  be  repeated  and  instead  of  rabbit  cells, 
red  cells  of  some  other  animal,  sheep,  for  instance,  be  added  to  the  heated 
haemolytic  serum  it  can  be  shown  that  the  sheep  cells  are  not  sensitized, 
since  on  the  addition  of  complement  they  are  not  haemolyzed. 

And  further,  the  serum  to  which  the  sheep  cells  have  been  added  has 
retained  intact  its  sensitizing  properties  and  is  still  capable  of  sensitizing 
rabbit  red  cells. 

This  experiment  again  demonstrates  the  specific  nature  of  the  reaction. 

(vi)  Haemolysis  does  not  occur  at  0°  C.  Place  a  mixture  of  non-heated 
hsemolytic  serum  and  a  suspension  of  the  corresponding  red  cells  in  the  ice 
chest  for  several  hours,  then  centrifuge  the  mixture  and  wash  the  cells  in 
the  cold,  add  some  complement  to  the  cells  and  incubate  at  37°  C.  :  haemo- 
lysis occurs.  Add  the  serum  to  some  sensitized  and  washed  red  cells  and  in 
this  case  also  haemolysis  occurs.  In  other  words  the  immune  body  has  been 
taken  out  of  the  serum  by  the  red  cells  but  ,at  the  temperature  of  the  experi- 
ment the  complement  remains  in  solution. 

Conclusions. 

When  a  living  animal  is  treated  with  sublethal  doses  of  micro-organisms 
or  their  toxins  (antigen)  a  substance  inimical  to  the  antigen  (antibody, 
amboceptor,  sensibilisatrice,  immune  body)  appears  in  the  serum  which  has 
the  property  of  combining  with  the  antigen,  thus  rendering  the  latter 
susceptible  to  the  action  of  a  third  substance  (complement,  alexin,  cytase) 
already  present  in  the  serum  of  the  normal  animal  and  derived  probably 
from  the  leucocytes. 

By  the  combined  action  of  the  immufte  body  and  complement,  the  antigen 
is  either  destroyed  (in  the  case  of  red  cells  or  delicate  organisms)  or  prepared 
for  the  destructive  action  of  the  leucocytes  (as  happens  with  micro-organisms 
in  general). 

The  fixation  of  the  complement. 

(Deviation  of  the  complement.) 

Prepare  in  accordance  with  the  rules  elaborated  in  the  preceding  para- 
graphs the  following  experiment. 

Mix  in  suitable  proportions  a  portion  of  a  culture  of  the  cholera  vibrio  and 
some  anticholera  serum  previously  heated  at  55°  C.,  incubate  for  1  hour,  and 
then  add  a  small  quantity  of  non-heated  serum  (complement)  to  the  mixture. 
Under  these  'conditions  the  vibrios  sensitized  by  the  specific  immune  serum 
are  bacteriolyzed  by  the  action  of  the  complement.  Now  add  to  the  mixture 
some  red  cells  sensitized  with  their  corresponding  inactivated  immune  serum 
(hcemolytic  couple) ;  no  haemolysis  takes  place  because  there  is  no  complement 
available,  the  complement  originally  present  having  all  been  used  up  in  pro- 
ducing bacteriolysis  of  the  cholera  vibrios.  In  other  words  there  has  been 
fixation,  or  deviation,  of  the  complement  by  the  sensitized  vibrios. 

Now  perform  a  second  experiment.  Mix  a  portion  of  a  culture  of  the 
typhoid  bacillus  with  some  inactivated  anticholera  immune  serum  and  after 
incubating,  add  a  small  quantity  of  guinea-pig  complement.  In  this  case, 
the  immune  body  has  not  been  able  to  sensitize  the  bacilli  being  specific  for 
and  combining  only  with  cholera  vibrios  :  consequently,  the  complement 
remains  unattached,  in  other  words,  is  not  deviated.  Now  add  some  sensitized 
red  cells  to  the  mixture  and  incubate  again ;  haemolysis  of  the  red  cells  occurs 
because  there  was  free  complement  in  the  mixture  with  which  they  were  able 
to  combine. 

From  this  fundamental  experiment  Bordet  and  Gengou  deduced  a  very 


FIXATION  OF  THE   COMPLEMENT  233 

valuable  method  of  diagnosis  for  infective  diseases  which  is  known  as  the 
Bordet-Gengou  or  complement-fixation  reaction.  The  reaction  has  been 
applied  by  Widal  and  Le  Sourd  to  the  diagnosis  of  enteric  fever  (fixation 
reaction,  hsemolyso-diagnosis)  and  is  applicable  to  the  majority  of  micro- 
organic  diseases.  Two  different  cases  arise  for  consideration. 

First  case. — Given  a  serum  suspected  to  contain  a  particular  immune  body, 
the  serum  of  an  enteric  fever  patient,  for  example,  a  certain  diagnosis  may 
be  made  by  the  complement-fixation  method. 

Heat  the  serum  to  55°  C.  for  half  an  hour,  prepare  a  mixture  of  typhoid 
bacilli  and  the  heated  serum,  add  some  complement  and  incubate  the  mixture 
at  37°  C.  for  an  hour.  Then  add  a  mixture  of  red  cells  and  homologous 
inactivated  hsemolytic  serum  and  incubate  again.  One  of  two  things  may 
happen. 

1.  Either  the  typhoid  bacillus  is  sensitized  by  the  inactivated  suspected 
serum,  in  which  case  it  fixes  the  complement  so  that  on  the  addition  of 
sensitized  red  cells — there  being  no  free  complement — the  cells  do  not  undergo 
haemolysis.     If  this  takes  place  it  may  be  affirmed  that  the  suspected  serum 
contains  antibodies  for  the  typhoid  bacillus  and  that  the  patient  is  suffering 
from  enteric  fever. 

2.  Or  the  typhoid  bacillus  is  not  sensitized  by  the  suspected  serum  and 
therefore  does  not  combine  with  the  complement,  so  that  on  the  addition  of 
sensitized  red  cells  the  free  complement  attaches  itself  to  them  and  haemolysis 
is  the  result.     The  suspected  serum,  therefore,  in  this  case  contains  no  typhoid 
antibodies. 

Second  case. — Suppose  a  given  organism  is  believed  to  be  the  typhoid 
bacillus  and  it  is  desired  to  confirm  the  diagnosis. 

Prepare  a  mixture  containing  the  suspected  bacillus,  heated  antityphoid 
serum  and  complement.  Incubate  for  an  hour  and  then  add  a  mixture  of 
red  cells  and  inactivated  haemolytic  serum. 

1.  The  bacillus  may  be  sensitized  by  the  antityphoid  serum  in  which  case 
it  will  absorb  the  complement,  and  on  the  addition  of  sensitized  red  cells — 
there  being  no  free  complement — no  haemolysis  takes  place  ;   the  complement 
was  deviated  or  fixed  by  the  suspected  bacillus  which  is  therefore  the  true 
typhoid  bacillus. 

2.  The  bacillus  may  not  be  sensitized  by  the  antityphoid  serum  conse- 
quently it  cannot  fix  the  complement  and  this  remaining  in  solution  is  free 
to  combine  with  the  sensitized  red  cells.     There  had  been  no  fixation  of  the 
complement  so  haemolysis  occurs  ;    the  bacillus  therefore,  not  uniting  with 
the  antityphoid  immune  body,  is  not  the  typhoid  bacillus. 

The  value  of  this  method  of  diagnosis  to  the  bacteriologist  can  be  easily 
appreciated  :  the  results  are  more  constant  and  more  delicate  than  those 
obtained  by  means  of  agglutination  tests  but  considerable  technical  skill  is 
required  in  carrying  out  the  reaction. 

Technique  of  the  complement-fixation  reaction. 

The  following  materials  are  required  : 

Apparatus,  etc. — 1.  A  number  of  narrow  test-tubes  about  10  cm.  long  and 
5  c.c.  capacity. 

2.  A  number  of  1  c.c.  pipettes  graduated  in  tenths  of  a  cubic  centimetre. 
(Levaditi's  pattern  is,  perhaps,  the  best  (fig.  160).) 

The  various  manipulations  should  as  far  as  possible  be  conducted  under 
aseptic  conditions,  so  that  the  tubes  and  pipettes  must  be  sterilized  in  the 
hot  air  sterilizer. 


234 


FIXATION  OF  THE  COMPLEMENT 


3.  Sterile  normal  saline  solution.  The  volume  of  fluid  in  each  tube  used  in 
the  test  should,  if  the  experiment  is  to  be  conclusive,  be  the  same.  After 
the  various  ingredients  have  been  added  sufficient  nor- 
mal saline  solution  is  poured  in  to  bring  the  volume  up 
to,  generally,  2  c.c. 

Red  cells. — Sheep  or  rabbit  red  cells  are  generally 
used.  They  must  be  separated  and  washed  in  the  manner 
already  described  (p.  230). 

After  the  third  washing  prepare  a  5  per  cent,  solution 
of  the  cells  in  normal  saline  solution. 

Haemolytic  serum. — The  method  of  preparing  haemo- 
lytic  serums  has  been  described  above  (p.  230).  If 
sheep  cells  are  used  as  the  indicator  the  serum  of  a 
rabbit  inoculated  with  sheep  cells  is  employed,  and 
for  rabbit  cells  the  serum  of  a  guinea-pig  inoculated  with 
rabbit  cells. 

After  collecting  the  blood  in  the  ordinary  way  (Chap. 
XII.)  the  serum  is  decanted  and  then  inactivated  by 
heating  for  half  an  hour  at  55°  C.  in  sealed  ampoules. 
The  hsemolytic  serum  should  be  stored  in  an  ice  chest 
and  it  will  then  retain  its  heemolytic  properties  for 
several  months. 

It  is  absolutely  necessary  to  titrate  the  haemolytic 
serum.  This  can  be  done  in  the  manner  indicated  in 
the  following  table.  In  a  series  of  tubes  prepare  the 
mixtures  shown  in  the  horizontal  lines,  incubate  at  37°  C.  for  15-30  minutes 
and  note  the  results. 


V 

100 

90 

- 

7 

80 
M 

1,8 

0,8 

70      K 

1.6          W 

Q. 

eo     E 

z 

Z 
1.+     1- 

M 

50       5 

z 
a 

JP 

40 

1.* 

0,4 

30 

2°     / 

I 

0,2 

" 

\l 

FIG.  160.—  Levadj 
pipettes. 

ti's 

5% 
emulsion 
of 
red  cells. 

Heated 
haemolytic 
serum. 

Comple- 
ment or 
alexin. 

Normal 
saline 
solution 
(to  make 
up  to 
2  c.c.). 

Results. 

Tube  No.  1,  - 

c.c. 
1 

c.c. 
0-5 

c.c. 

o-i 

c.c. 
0-4 

Total  haemolysis  in 
15  minutes. 

Tube  No.  2,  - 

1 

0-3 

o-i 

0-6 

Total  haemolysis  in 
15  minutes. 

Tube  No.  3,  - 

1 

0-1 

o-i 

0-8 

Partial  haemolysis  in 
15  minutes,   com- 
plete in  30  minutes. 

Tube  No.  4,  - 

1 

0-05 

o-i 

0-85 

Incomplete  haemolysis 

Tube  No.  5,  - 

1 

o-oi 

o-i 

0-9 

Very  slight  haemolysis. 

Control  No.  1, 

1 

— 

O'l 

0-9 

No  haemolysis. 

Control  No.  2, 

1 

0-5 

— 

0-5 

No  haemolysis. 

FIXATION   OF  THE  COMPLEMENT 


235 


From  an  examination  of  the  table  it  follows  that  the  amount  of  haemolytic 
serum  added  to  tube  No.  3  will,  in  this  particular  instance,  be  the  most 
suitable  for  subsequent  experiments  :  in  this  tube  haemolysis  is  complete 
in  half  an  hour.  Tubes  Nos.  1  and  2  contain  too  much  serum,  and  in  Nos.  4 
and  5  haemolysis  is  not  complete  and  they  therefore  contain  too  little  serum. 

Further,  examination  of  the  control  tubes  shows  : 

1.  That  the  heated  haemolytic  serum  only  haemolyzes  when  complement 
is  added. 

2.  That  complement  alone  does  not  haemolyze  the  red  cells. 

In  carrying  out  the  reaction  of  complement  fixation  then,  the  quantity  of 
Ol  c.c.  of  this  particular  hsemolytic  serum  per  1  c.c.  of  the  dilution  of  red 
cells  will  be  used. 

Complement. — Normal  guinea-pig  serum  collected  aseptically  will  be  used 
as  complement.  The  amount  of  complement  to  be  added  is  of  the  greatest 
importance  :  if  there  be  an  excess  of  complement  the  whole  of  it  will  not  be 
absorbed  by  the  antigen-antibody  mixture  and  the  excess  remaining  in 
solution  will  haemolyze  the  red  cell-serum  mixture  and  give  an  erroneous 
result.  The  smallest  quantity  of  guinea-pig  serum  which  will  haemolyze 
1  c.c.  of  the  sensitized  red  cell  emulsion  must  therefore  be  determined. 

If  fresh  guinea-pig  serum  be  used  it  will  be  found  to  be  very  rich  in  com- 
plement but  the  amount  rapidly  diminishes  in  the  first  few  hours.  It  is 
preferable  therefore  to  use  serum  collected  8-10  days  before  [and  stored  in  an 
ice  chest] ;  it  is  not  so  active  but  its  titre  remains  constant  for  several  days 
(Nicolle  and  Pozerski).  The  following  table  illustrates  the  method  of  titration. 
Incubate  the  tubes  for  half  an  hour  at  37°  C.  and  then  note  the  results. 


5% 
emulsion 
of 
red  cells 
in  normal 

Heated 
hsemolytic 
serum. 

Comple- 
ment 
(normal 
guinea-pig 
serum 

Normal 
saline 
solution 
to  make 

Results. 

. 

saline 
solution. 

8  days 
old). 

2  c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

Tube  No.  1,  - 

1 

o-i 

0-2 

0-7 

} 

Tube  No.  2,  - 

1 

o-i 

o-i 

0-8 

>  Complete  haemolysis. 

Tube  No.  3,  - 

1 

o-i 

0-05 

0-85 

J 

Tube  No.  4,  - 

1 

o-i 

0-03 

0-9 

) 

^Incomplete  haemolysis. 

Tube  No.  5,  - 

1 

o-i 

0-02 

0-9 

J 

Tubes  Nos.  1,  2  and  3  alone  contain  enough  complement  to  produce  com- 
plete haemolysis.  The  quantity  in  tube  3  will  be  used  because  it  is  the 
smallest  quantity  which  produces  complete  haemolysis. 

Antigen. — The  organisms  to  be  used  as  antigen  should  be  prepared  as 
follows  :  Take  a  young  agar  culture  (in  the  case  of  the  cholera  vibrio  or 
typhoid  bacillus,  for  instance,  a  24  or  48-hour  culture)  and  emulsify  one 
loopful  in  2  c.c.  of  sterile  normal  saline  solution.  In  carrying  out  the  experi- 
ment small  quantities  only  of  this  emulsion  are  used  because  large  quantities 
of  albuminoid  substances  may  produce,  in  the  absence  of  a  specific  reaction, 
a  mechanical  deviation  of  the  complement  and  so  give  fallacious  results. 


236 


FIXATION  OF  THE  COMPLEMENT 


The  bacillary  emulsion  should  be  titrated  by  placing  in  the  incubator  at 
37°  C.  a  series  of  tubes  containing  progressively  increasing  quantities  of  the 
emulsion,  an  haemolytic  couple  (p.  232)  and  some  complement. 


Emulsion 
of 
organisms. 

Complement. 

Emulsion 
of 
red  cells. 

Heated 
hsemolytic 
.  serum. 

Normal 
saline 
solution. 

Results. 

c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

Tube  No.  1, 

0-05 

0-05 

1 

o-i 

0-80 

^ 

Tube  No.  2, 
Tube  No.  3, 

o-i 

0-2 

0-05 
0-05 

1 

1 

o-i 
o-i 

0-75 
0-65 

Complete 
haemolysis. 

Tube  No.  4, 

0-3 

0-05 

1 

o-i 

0-55 

_, 

Tube  No.  5, 
Tube  No.  6, 

0-4 
0-5 

0-05 
0-05 

1 
1 

o-i 
o-i 

0-45 
0-35 

1  Incomplete 
1     haemolysis. 

Examination  of  the  table  shows  that  the  quantity  of  bacterial  emulsion 
to  be  used  must  be  the  amount  contained  in  tube  3  or  tube  4,  namely  0*2 
or  0'3  c.c.  these  being  the  maximum  doses  which  do  not  prevent  haemolysis 
taking  place. 

In  the  serum  diagnosis  of  syphilis,  as  it  is  not  possible  to  obtain  a  culture 
of  the  treponeme,  various  other  substances  are  used  as  the  antigen,  e.g.  an 
extract  of  the  liver  of  a  syphilitic  foetus.  This  will  be  referred  to  later 
(vide  Chap.  LIV.). 

Bacteriolytic  serums. — Bacteriolytic  serums  are  obtained  either  from 
immunized  animals  or  from  man.  A  small  quantity  of  blood — 4  to  5  c.c. — is 
sufficient  and  may  be  obtained  in  the  case  of  the  human  subject  by  puncture 
of  a  vein  at  the  bend  of  the  elbow  or  with  the  aid  of  a  Bier's  cupping  glass  ; 
in  the  case  of  the  rabbit  by  puncturing  an  ear  vein  (p.  194).  After  being 
collected  the  blood  is  put  aside  for  a  few  hours,  and  the  serum  is  then  pipetted 
off  and  heated  in  sealed  tubes  at  55°  C.  for  half  an  hour  to  destroy  the  com- 
plement. In  carrying  out  the  experiment  the  bacteriolytic  serum  should 
be  added  to  the  emulsion  of  bacteria  in  sufficient  quantity  to  sensitize  them 
but  the  actual  amount  required  for  sensitization  should  not  be  greatly  exceeded 
for  fear  of  introducing  errors.  The  serum  can  be  titrated  by  a  method  similar 
to  that  used  for  titrating  the  antigen  (vide  ante). 

Experimental  details. — The  reagents  being  prepared,  assume  that  it  is 
desired  to  determine  whether  a  given  vibrio  is  the  cholera  vibrio  or  not. 
The  experiment  will  be  carried  out  as  shown  in  the  table  on  p.  237. 

In  this  experiment  no  haemolysis  has  taken  place  in  the  tubes  Nos.  2,  3,  4  : 
therefore  the  vibrio  under  investigation  was  sensitized  by  the  cholera  immune 
serum  and  was  able  to  combine  with  the  complement.  The  organism,  there- 
fore, is  the  cholera  vibrio. 

In  tube  No.  1  the  quantity  of  vibrio  emulsion  was  not  quite  sufficient,  and 
this  is  the  reason  why  the  fixation  has  not  been  complete. 

Examination  of  the  control  tubes  confirms  the  diagnosis  by  proving  that 
in  the  absence  of  anticholera  serum  in  one  case  and  in  the  absence  of  the 
cholera  vibrio  in  the  other  no  fixation  has  occurred,  and  therefore  the  tubes 
show  haemolysis. 


FIXATION  OF  THE   COMPLEMENT 


237 


r[f,  however,  the  results  had  been  as  follows  : 
Tubes  Nos.  1,  2,  and  3  ==  complete  haemolysis. 
Tube  No.  4  = slight  haemolysis. 

it  would  have  been  concluded  that  the  vibrio  had  not  been  sensitized  by  the 
cholera  immune  serum  ;  that  consequently  there  was  no  fixation  of  the 
complement ;  and  that  therefore  the  vibrio  could  not  have  been  the  cholera 
vibrio.  The  assumed  occurrence  of  partial  haemolysis  in  tube  No.  4  is  to  be 
explained  as  due  to  a  slight  excess  of  bacterial  emulsion ;  the  micro 
organisms  alone  having  absorbed  some  of  the  complement  in  the  manner 
already  described  (p.  235). 


DETAILS  OF  A  COMPLEMENT-FIXATION  EXPERIMENT  AS  ARRANGED  FOR  THE 

IDENTIFICATION   OF  A   SUSPECTED   CHOLERA   VIBRIO   (see  p.    236). 


(i)  Mix  and  incubate  for 
one  hour  at  37°  C. 

(ii)  Add  at  the  end  of 
the  hour  and  incu- 
bate again  for  half 
an  hour  at  37°  C. 

Results. 

Emulsion 
of 
vibrios. 

Heated 
anti- 
cholera 
serum. 

Complement. 

Normal 
saline 
solution. 

Emulsion 
of 
red  cells. 

Heated 
hsemolytic 
serum. 

Tube  No.  1, 

c.c. 
O'l 

c.c. 
O'l 

c.c. 
0-05 

c.c. 
0-65 

c.c. 
1 

c.c. 

o-i 

/  Slight 
i.   haemolysis. 

Tube  No.  2, 

0-2 

o-i 

0-05      j       0-55 

1 

o-i 

V 

Tube  No.  3, 

0-3 

o-i 

0-05 

0-45 

1 

o-i 

[  No  hsemo- 
1     lysis. 

Tube  No.  4, 

0-4 

o-i 

0-05 

0-35 

1 

o-i 

J 

Control,     - 
Control,     - 

o-i 

02 



0-05 
0-05 

0-75 
0-65 

1 
1 

o-i 
o-i 

1  Complete 
f    haemolysis. 

Control,     - 

— 

o-i 

0-05 

0-75 

1 

o-i 

/  Complete 
t    haemolysis. 

Control, 

— 

— 

o-i 

0-80 

1 

o-i 

f  Complete 
I    haemolysis. 

To  put  the  result  beyond  all  doubt  it  is  still  necessary  to  show : 

1.  That  the  cholera  serum  in  the  quantity  in  which  it  was  used  does  not 
fix  the  complement  in  presence  of  any  other  species  of  bacterium,  and  in  an 
actual  experiment  an  additional  control  tube  would  have  been  introduced 
containing  instead  of  the  vibrio  emulsion  an  emulsion  of,  for  instance,  the 
typhoid  bacillus.     Haemolysis  should,  of  course,  occur  under  these  conditions. 

2.  That  the  vibrio  under  investigation  does  not  fix  the  complement  in 
presence  of  another  serum.    Another  control  tube  would  therefore  be  prepared 
with  the  emulsion  of  the  vibrio  but  substituting,  for  example,  an  antityphoid 
serum  for  the  anticholera  serum.     Here  again  haemolysis  should  take  place. 

The  above  then  is  the  method  of  applying  the  complement  fixation  reaction 
to  the  identification  of  an  unknown  organism.     The  data  can  be  reversed 


238 


FIXATION  OF  THE   COMPLEMENT 


and  the  reaction  applied  to  determine  whether  a  given  serum  contains  anti- 
bodies for  a  given  organism.  In  illustration,  an  example  will  now  be  given 
to  show  how  to  determine  whether  or  no  the  serum  of  a  patient  contain 
typhoid  antibodies  (hsemolyso -reaction  of  Widal  and  Le  Sourd). 

In  this  case  the  suspected  serum  after  heating  at  55°  C.  is  mixed  with  a 
known  typhoid  bacillus  and  complement.  The  experiment  is  arranged  in 
the  same  way  as  in  the  preceding  experiment. 

DETAILS  OF  A  COMPLEMENT-FIXATION  EXPERIMENT  AS  ARRANGED  FOR  THE 
IDENTIFICATION  OF  A  SUSPECTED  ENTERIC  SERUM. 


(i)  Mix  and  incubate  for 
one  hour  at  37°  C. 

(ii)  Add  at  the  end  of 
the  hour  and  incu- 
bate again  for  half 
an  hour  at37°C. 

Results. 

Emulsion 
of 
typhoid 
bacilli. 

Heated 
suspected 
serum. 

Complement. 

Normal 
saline 
solution. 

Emulsion 
of 
red  cells. 

Heated 
haemolytic 
serum. 

Tube  No.  1, 
Tube  No.  2, 

c.c. 
0-2 

0-3 

c.c. 
O'l 

0'3 

c.c. 
0-05 

0-05 

c.c. 
0-55 

0-25 

c.c. 
1 

1 

c.c. 

o-i 
o-i 

1  No  haemo- 
f    lysis. 

Control,     - 

0-3 

Normal 
human 
serum. 

0-3 

0-05 

0-25 

1 

o-i 

/  Complete 
I   haemolysis. 

Control,      - 

0-3 

— 

0-05 

0-55 

1 

o-i 

(  Complete 
\    haemolysis. 

Control, 

— 

— 

0-05 

1 

0-85              1 

o-i 

f  Complete 
\   haemolysis. 

In  tubes  Nos.  1,  and  2  no  haemolysis  has  occurred  ;  the  typhoid  bacillus 
it  is  evident  has  been  sensitized  by  the  serum  under  examination,  which 
must  therefore  have  come  from  a  patient  infected  with  the  typhoid  bacillus. 

Examination  of  the  control  tubes  shows  that  the  bacillus  alone  or  in  presence 
of  normal  human  serum  is  unable  to  fix  the  complement  with  the  result  that 
haemolysis  has  taken  place. 

If,  on  the  other  hand,  the  serum  under  investigation  had  not  sensitized  the 
typhoid  bacillus  haemolysis  would  have  occurred,  and  the  inference  would 
have  been  that  the  serum  contained  no  typhoid  antibodies. 

Practical  applications. 

The  method  of  complement  fixation  has  been  applied  to  the  diagnosis  of  a 
large  number  of  micro-organic  diseases  (enteric  fever,  cholera,  dysentery, 
tuberculosis,  etc.)  and  to  the  identification  of  most  micro-organisms.  It  is 
also  the  basis  of  Wassermann's  reaction  in  syphilis  which  will  be  considered 
later  (Chap.  LIV.). 


FIXATION  OF  THE   COMPLEMENT  239 


SECTION  V.— OPSONINS. 

In  studying  the  bactericidal  properties  of  serums  it  has  been  mentioned 
that  many  micro-organisms  resist,  in  vitro,  the  combined  action  of  immune 
body  and  complement,  but  that  in  the  tissues  once  impregnated  with 
these  substances  they  more  readily  become  the  prey  of  the  phagocytes 
(MetchnikofE). 

Wright  and  Douglas  have  shown  that  in  the  serum  of  persons  convalescent 
from  infectious  diseases  or  vaccinated  against  these  diseases  substances  are 
present  which  prepare  micro-organisms  for  the  action  of  the  phagocytes. 
Without  committing  themselves  to  an  expression  of  opinion  as  to  the  nature 
of  these  substances  Wright  and  Douglas  describe  them  as  opsonins  (oi/'wi/ew 
I  prepare).  Neufeld  has  applied  to  them  the  name  Bacteriotropins. 

According  to  Wright,  opsonins  play  a  fundamental  part  in  the  phenomena 
of  immunity  :  he  affirms  that  it  is  to  opsonins  that  phagocytosis  is  due  and 
that  by  means  of  the  opsonic  index  of  the  serum  it  is  possible  to  measure  the 
immunity  of  the  individual  and  foresee  recovery. 

Metchnikofif  has  observed  that,  as  a  matter  of  fact,  the  ingestion  of  micro-organisms 
by  phagocytes  rendered  possible  by  the  intervention  of  opsonins  is  only  one  factor 
in  the  problem.  Ingestion  is  only  of  use  in  so  far  as  it  is  followed  by  destruction 
and  digestion  of  the  organisms.  But  micro-organisms  are  not  destroyed  by  leucocytes 
unless  the  latter  contain  bactericidal  substances  or  in  other  words  unless  the  leuco- 
cytes are  "  living  and  strong."  Resistant  micro-organisms  may  live  for  a  long 
time  in  insufficiently  active  leucocytes  without  setting  up  disease  but  when  such 
leucocytes  are  destroyed  the  micro-organisms  are  set  free  and  exhibit  their  powers 
of  producing  disease.  A  notable  instance  of  this  is  seen  in  the  case  of  the  spores 
of  the  tetanus  bacillus  (Chap.  XXXVI.).  The  opsonic  content  of  the  serum  is  not 
therefore — at  any  rate  in  all  cases — a  sufficient  datum  upon  which  to  evaluate  the 
degree  of  resistance  of  the  tissues. 

However  that  may  be,  opsonins  are  of  sufficient  interest  in  the  study  of  micro- 
organic  diseases  and  immunizing  processes  to  merit  some  detailed  consideration. 

To  determine  the  opsonic  content  of  a  given  serum  for  a  particular  micro- 
organism, the  serum  and  a  culture  of  the  organism  are  mixed  with  normal 
leucocytes  and  after  an  interval  the  average  number  of  micro-organisms 
ingested  by  each  leucocyte  under  these  conditions  calculated.  The  number 
of  organisms  ingested  by  50  leucocytes  is  counted  and  the  total  divided  by 
50  gives  the  opsonic  power  of  the  serum. 

It  is  obvious,  of  course,  that  the  number  of  organisms  phagocyted  will 
depend  upon  the  number  of  bacteria  present  in  a  unit  volume  of  the  emulsion. 
The  absolute  number  obtained — i.e.  the  opsonic  power — is  therefore  of  no 
value  in  itself,  but  if  this  number  be  compared  with  the  number  which 
represents  the  opsonic  power  of  a  normal  serum  determined  under  the  same 
conditions  with  the  same  bacterial  emulsion  then  a  standard  of  comparison 
is  obtained  ;  and  the  relation  of  the  opsonic  power  of  the  serum  of  an  infected 
individual  to  that  of  a  normal  individual  (measured  under  identical  con- 
ditions) is  known  as  the  opsonic  index. 

The  amount  of  opsonin  present  in  the  serum  of  normal  individuals  is  subject 
to  considerable  variation  and  is  dependent  upon  many  factors,  e.g.  the  period 
which  has  elapsed  since  food  was  last  taken,  pregnancy,  etc.  (Milhit). 

The  amount  of  specific  opsonin  in  the  serum  of  infected  persons  shows  very 
curious  variation.  In  tuberculosis,  for  example,  if  the  opsonic  index  of  normal 
blood  for  the  tubercle  bacillus  be  taken  to  be  about  unity  that  of  infected 
persons  is  much  lower,  and  a  condition  of  tuberculosis  may  be  diagnosed  in 
every  case  in  which  the  opsonic  index  falls  below  unity  (0'3  to  0'8),  provided 
that  the  experiment  be  done  several  times  and  the  same  result  is  obtained 


240 


OPSONINS 


on  every  occasion.     The  reaction  is  much  more  reliable  in  the   "  surgical 

tuberculoses"  than  in  pulmonary  tuberculosis. 

In  suspected  cases  of  enteric  fever  an  opsonic  index  above  1*7  for  the 

typhoid  bacillus  affords  strong  presumptive  evidence  in  favour  of  enteric 

fever  (Milhit).     Similarly  in  cerebro-spinal  meningitis  the  opsonic  index  is 

raised    above    normal    during    the 
course  of  the  disease  ;   and  so  on. 

Method  of  determining  the  opsonic 
index. 

The  following  materials  and  appa- 
ratus are  necessary  : 

Apparatus. —  Small  test-tubes  — 
Pasteur  pipettes  fitted  with  india- 
rubber  teats,  these  should  be  made 
from  tubing  4-5  mm.  in  diameter 
and  be  drawn  out  rather  long  (fig. 
162).— Bulb  pipettes  fitted  with  a 
small  india-rubber  aspirating  tube. 
—  A  mechanical  centrifuge.  —  An 
Hearson's  opsonic  incubator  (fig. 
161)  heated  by  gas  or  electricity 

FIG.  161.— Opsonic  incubator.  an(J  regulated  at  37°  C. 

A  supply  of  sterile  normal  saline  solution  and  of  citrated  saline  solution 
must  be  at  hand  : — 

Sodium  chloride,     -  8 '5  grams. 

Sodium  citrate,        -  15  „ 

Distilled  water,        -  -        Q.S.  ad  1  litre. 

Normal  serum. — Collect  some  blood  in  a  small  centrifuge  tube  either  from 
an  animal  by  puncture  of  an  ear  vein  or  from  man  by  pricking  the  finger. 
(Chap.  XII.).  Centrifuge  the  blood  at  once  and  decant  the  serum  with  a 
Pasteur  pipette.  The  serum  is  best  collected  when  the  individual  is  fasting 
(Milhit) :  it  should  be  used  as  soon  as  possible  and  always  within  2  or  3  hours 
of  collection  because  the  opsonic  power  rapidly  diminishes. 

Patient's  serum. — This  should  be  collected  under  the  same  conditions  and  at  the 
same  time  as  the  normal  serum  in  order  that  the  results  may  be  comparable. 

Bacterial  emulsion. — [When  the  rate  of  growth  permits]  very  young 
cultures  should  be  used  (8-24  hours  old  according  to  the  organism),  and  for 
the  same  series  of  experiments  cultures  of  the  same  age  and  prepared  under 
identical  conditions  are  necessary. 

As  a  rule,  agar  cultures  are  used  and  one  loopful  is  made  into  an  emulsion 
with  2  c.c.  of  normal  saline  solution.  The  emulsion  must  be  carefully  pre- 
pared ;  it  should  be  slightly  opalescent  when  held  up  to  the  light  and  must 
be  perfectly  homogeneous.  If  too  thick  an  emulsion  be  used  it  will  be  difficult 
to  enumerate  the  organisms  in  the  subsequent  part  of  the  experiment. 

Leucocytes.— 1.  Cleanse  the  skin  of  the  thumb,  tie  an  india-rubber  ligature 
around  its  proximal  end  and  then  make  several  little  pricks  on  the  dorsal 
surface  of  the  distal  end  near  the  root  of  the  nail. 

2.  Allow  about  thirty  drops  of  blood  to  flow  into  a  small  sterile  centrifuge 
tube  containing  about  10  c.c.  of  citrated  normal  saline  solution  (fig.  147,  p.  192). 

3.  Mix  the  blood  and  citrate  solution  carefully,  centrifuge  the  mixture  for 
15  minutes,  decant  the  supernatant  liquid  with  a  bulb  pipette,  add  an  equal 
volume  of  normal  saline  solution  to  the  deposit,  mix  and  centrifuge  again. 
Then  decant  the  saline  solution  and  wash  a  second  time. 


OPSONINS  241 


4.  After  washing  three  times  decant  the  supernatant  liquid  being  careful 
not  to  stir  up  the  deposit  of  cells.  Lay  the  tube  as  nearly  horizontally  as 
possible  and  leave  it  for  about  half  an  hour.  Then  collect  the  upper  whitish 
layer  of  cells  which  is  composed  almost  exclusively  of  leucocytes.  The  leuco- 
cytes ought  to  be  used  within  6  hours  of  the  blood  being  collected  (Milhit). 

Experimental  details. 

1.  Take  a  Pasteur  pipette  ready  furnished  with  an  india-rubber  teat,  cut 
off  the  capillary  end  squarely  with  a  carburundum  pencil  and  make  a  small 
mark  on  the  glass  about  2  cm.  from  the  point. 


FIG.  162. — Preparation  of  the  mixture  for  the  determination  of  the  opsonic 
index.  The  figure  shows  the  three  equal  volumes  of  fluid  aspirated  into  the 
pipette  and  separated  by  two  bubbles  of  air. 

2.  Aspirate  into  the  pipette  in  turn  by  lightly  relaxing  the  teat : 

(a)  A  column  of  leucocytes  up  to  the  mark  on  the  glass,  then  a  small 
bubble  of  air. 

(6)  A  column  of  bacterial  emulsion  up  to  the  same  mark,  then  another 
bubble  of  air. 

(c)  A  column  of  the  serum  to  be  examined,  again  up  to  the  mark. 

There  are  now  three  equal  volumes  of  fluid  in  the  pipette  separated  by  two 
small  bubbles  of  air  (fig.  162). 

3.  Expel  the  liquids  on  to  a  sterile  slide  and  mix  them  together,  then 
draw  up  the  mixture  into  the  pipette  again,  being  careful  to  avoid  taking  up 
any  air  bubbles.     Seal  the  end  in  the  pilot  of  a  Bunsen,  and  place  the  pipette 
horizontally  in  the  opsonic  incubator  at  37°  C.  for  15  minutes. 

4.  Break  off  the  end  of  the  pipette.     Place  a  drop  of  the  mixture  on  each 
of  several  slides,  spread  rapidly,  dry  and  fix  the  films  by  heat  or  alcohol- 
ether.     Stain  with  carbol-thionin  or  in  the  case  of  the  tubercle  bacillus  with 
carbol-fuchsin. 

5.  With  an  oil-immersion  lens  count  the  number  of  the  organisms  phagocy  ted 
by  50  leucocytes. 

For  example,  120  organisms  are  counted  in  50  leucocytes  :  the  opsonic 
power  of  the  serum  examined  is  therefore  --?£-  =  2*4:0. 

6.  Repeat  the  experiment  using  normal  serum.     Suppose  90  organisms 
are  counted  in  50  leucocytes :  the  opsonic  power  of  the  normal  serum  is 

»  o  _  1  .of\ 
5(7  —  1  OU. 

(For  clearness  of  description  the  two  investigations— the  opsonic  power  of 
the  suspected  serum  and  that  of  the  normal  serum — have  been  described 
successively.  In  practice,  of  course,  they  will  be  taken  in  hand  together.) 

7.  The  opsonic  index  being  the  ratio  of  the  opsonic  power  of  the  suspected 
serum  to  that  of  the  normal  serum  is  : 

2-40    _  1.QO 
1-80   ~  1  °°* 


PART  II. 
THE   BACTEKIA. 


CHAPTER   XV. 
BACILLUS  DIPHTHERIA.1 

Introduction. 

Section  I. — Experimental  inoculation,  p.  247. 

1.  Symptoms  and  lesions  produced  in  animals  susceptible  to  infection,  p.  247. 
2.  Influence  of  other  organisms  on  the  clinical  course  of  the  disease,  p   249. 
Section  II. — Morphology,  p.  250. 

1.  Microscopical  appearance  and  staining  reactions,  p.  250.     2.  Cultural  charac- 
teristics, p.  253. 
Section  III. — Biological  properties,  p.  254. 

1.  Vitality  and  virulence,  p.  254.  2.  Bio-chemical  reactions,  p.  256.  3.  Toxin, 
p.  257.  4.  Vaccination,  p.  262.  5.  Serum  therapeutics,  p.  265.  6.  Agglutination, 
p.  269. 

Section    IV. — Detection,    isolation    and    identification   of   the    diphtheria    bacillus — The 
clinical  diagnosis  of  diphtheria,  p.  269. 

1.  Collection  of  the  material  p.  270.  2.  Methods  of  examination,  p.  270.  3.  Sum- 
mary of  diagnostic  tests,  p.  273. 

Bacillus  pseudo-diphthense. 

(Hofmann's  bacillus). 

1.  Introduction,  p.  273.  2.  Morphology  and  staining  reactions,  p.  273.  3.  Cultural 
characteristics  and  bio-chemical  reactions,  p.  274.  4.  Virulence  and  immunity 
reactions,  p.  274.  5.  The  relation  of  Hofmann's  bacillus  to  the  diphtheria  bacillus, 
p.  274. 

THE  diphtheria  bacillus  was  discovered  by  Klebs  but  the  first  complete 
description  of  the  organism  was  contributed  by  Lceffler,  while  the  specific 
relationship  of  the  bacillus  to  the  disease  was  established  by  Roux  and  Yersin 
who  experimentally  produced  symptoms  of  paralysis  in  animals. 

Distribution  of  the  diphtheria  bacillus. 

1.  In  man. 

The  Klebs-Lceffler  bacillus  is  found  in  the  false  membranes  of  faucial,  nasal  and 
cutaneous  diphtheria,  and  in  croup.  Inflammatory  conditions  of  the  throat  in 
which  no  false  membrane  is  formed  are  also  sometimes  due  to  the  diphtheria  bacillus, 
and  in  these  cases  the  true  nature  of  the  disease  can  only  be  determined  by  bacterio- 
logical examination. 

The  bacillus  is  generally  localized  in  the  false  membrane  or  on  the  infected  mucous 
membrane  :  it  does  not  as  a  rule  invade  the  tissues  :  death  is  the  result  of  a  true 

1  The  diphtheria  bacillus  with  the  pseudo-diphtheria  bacillus,  the  xerosis  bacillus  and 
the  bacillus  of  glanders,  are  by  German  writers  classified  together  as  the  Corynebacteria, 
and  known  respectively  as  the  C.  diphtheria,  C.  commune,  C.  conjunctive  and  C.  mallei. 
The  group  is  characterized  by  the  presence  of  metachromatic  granules  and  club-shaped 
swellings  at  the  ends  of  the  organisms,  and  by  the  appearance  of  branched  forms  in  old 
cultures. 


246  THE  DIPHTHERIA  BACILLUS 

intoxication.  In  a  few  severe  cases  of  diphtheria  however  the  organism  has  been 
found  after  death  in  the  blood  and  internal  organs  (Babes,  Spronck,  and  others) : 
and  it  is  frequently  found  in  the  broncho- pneumonic  patches  which  follow  an  attack 
of  croup  (Lceffler,  Kutscher). 

The  bacillus  is  also  found  in  the  mouths  and  nasal  cavities  of  persons  who  have 
suffered  from  diphtheria,  sometimes  for  many  weeks  after  recovery  from  the  disease. 
["  In  3  weeks  about  30  per  cent,  of  diphtheria  patients  are  free  from  morphologically 
typical  diphtheria  bacilli.  In  20  per  cent,  the  bacilli  persist  for  4  weeks,  in  16  per 
cent,  for  5  weeks,  and  in  11  per  cent,  for  7  weeks.  One  per  cent,  harbour  them 
for  15  weeks  and  in  exceptional  cases  they  remain  in  the  throat  for  30  weeks,  though 
even  more  prolonged  periods  of  persistence  are  recorded  "  (Graham-Smith).  Fully 
virulent  diphtheria  bacilli  have  been  recovered  after  as  long  as  335  days  (Prip), 
230  days  (Schafer),  215  days  (Belfanti) :  these  and  other  observations  "  conclusively 
prove  that  diphtheria  bacilli  are  capable  of  retaining  their  virulence  during  very 
prolonged  persistence  in  the  throats  of  infected  persons  "  (Graham-Smith).  ]l 

[Diphtheria  bacilli,  a  very  large  proportion  of  which  are  virulent,  are  also  present 
in  the  throats  and  noses  of  "  contacts  " — persons  who  have  recently  been  in  intimate 
connexion  with  the  disease.  It  would  even  appear  that  less  than  half  the  number 
of  individuals  in  whom  the  bacillus  obtains  a  lodgment  are  attacked  by  the  disease. 
Graham-Smith  gives  statistics  which  show  that  amongst  infected  families  (relatives 
and  attendants)  36*6  per  cent,  are  liable  to  become  infected,  while  the  mean  per- 
centage of  infection  amongst  inmates  of  hospital  wards  and  institutions  is  14  per 
cent,  "and  amongst  scholars  of  infected  schools  8*7  per  cent.  ] 

Though  the  fact  is  denied  by  several  writers  there  can  be  no  doubt  but  that  the 
diphtheria  bacillus  may  occasionally  be  found  in  the  mouths  of  persons  who 
have  not  been  in  contact  with  diphtheria  :  [but  an  investigation  in  England  showed 
that  of  2132  persons  who  had  not  so  far  as  could  be  determined  been  exposed  to  in- 
fection 0'18  per  cent,  were  found  to  be  harbouring  a  virulent  diphtheria  bacillus  and 
2 '62  per  cent,  non- virulent  bacilli,  and  in  the  absence  of  further  evidence  these 
figures  undoubtedly  point  to  the  conclusion  "that  virulent  diphtheria  bacilli  are 
seldom  if  ever  present  in  the  throats  of  healthy  persons  who  have  not 
recently  been  in  contact  with  cases  of  diphtheria  or  infected  contacts  "  (Graham- 
Smith).] 

2.  In  the  lower  animals. 

[Cows. — According  to  Klein  cows  can  be  experimentally  infected  with  diphtheria, 
and  lesions  containing  diphtheria  bacilli  may  appear  on  the  teats  and  udders  as  a 
result  of  the  infection :  diphtheria  bacilli  may  also  be  present  in  the  milk  after 
experimental  inoculation.  From  these  observations  Klein  inferred  that  cows  might 
naturally  suffer  from  diphtheria  and  that  the  milk  of  such  cows  might  be  a  cause 
of  human  infection. 

[Klein's  experimental  results  have  not  been  confirmed  and  most  observers  hold 
that  there  is  no  evidence  that  diphtheria  is  a  bovine  disease  (Graham-Smith). 
On  two  occasions  however  virulent  diphtheria  bacilli  have  been  recovered  from  spon- 
taneous lesions  of  the  udder  and  teats  of  cows.  In  one  of  these  cases  investigated 
by  Dean  and  Todd  these  observers  came  to  the  conclusion  that  though  diphtheria 
bacilli  were  present,  the  lesions  in  the  cow  were  not  due  to  that  organism.  In  the 
other  case  Dean  and  M'Conkey  independently  isolated  the  diphtheria  bacillus  from 
the  lesions  in  the  cow  but  neither  the  source  of  the  bacillus  nor  its  relation  to  the 
ulcers  was  determined. 

[Horses. — The  diphtheria  bacillus  has  only  once  been  isolated  from  the  horse.  It 
was  then  found  by  Cobbett  in  a  purulent  and  slightly  sanguineous  discharge  from 
the  nose  of  a  pony. 

[Cats  and  fowls  and  other  animals. — "  Both  cats  and  fowls  have  frequently  been 
regarded  as  carriers  of  the  disease,  but  the  bacteriological  evidence  in  support  of 
these  statements  is  unsatisfactory.  Instances  of  natural  infection  amongst  other 
animals  are  unknown,  though  bacilli  closely  resembling  diphtheria  bacilli  in  many 
of  their  characters  have  been  found  in  dogs,  guinea-pigs,  rats,  fowls,  turkeys  and 
pigeons  "  (Graham-Smith). 

[These  observations  on  the  occurrence  of  diphtheria  in  the  lower  animals  may  be 

1  The  Bacteriology  of  Diphtheria,  edited  by  G.  H.  F.  Nuttall  and  G.  S.  Graham-Smith ; 
Camb.  Univ.  Press. 


sum 


EXPERIMENTAL  INOCULATION  247 


summarized  by  saying  that  only  once  has  a  true  diphtheria  bacillus  been  isolated 
from  an  animal  spontaneously  infected.  The  disease  must  therefore  be  exceedingly 
uncommon  among  the  lower  animals,  and  statements  to  the  effect  that  a  case  has 
been  observed,  or  an  outbreak  of  diphtheria  traced  to  infection  from  the  lower 
animals,  should  not  be  accepted  without  rigorous  investigation.] 

3.  In  the  circumambient  media. 

The  diphtheria  bacillus  is  able  to  live  outside  the  human  body.  Park,  Wright  and 
Emerson  found  the  organism  in  the  dust  of  diphtheria  wards  and  on  the  clothes  of 
the  attendants.  Abel  also  found  it  on  the  toys  with  which  children  infected  with 
diphtheria  had  been  playing. 

Bacillus  pseudo-diphtherise. 

The  pseudo-diphtheria  or  Hofmann's  bacillus,  an  organism  in  some  respects  closely 
resembling  the  diphtheria  bacillus  but  differing  from  it  in  being  shorter  and  non- 
pathogenic  to  laboratory  animals  and  in  other  particulars,  is  fairly  frequently  met 
with  in  the  mouths  of  healthy  persons. 

Reference  is  again  made  to  this  organism  later  in  the  present  chapter  (p.  273). 


SECTION   I.— EXPERIMENTAL  INOCULATION. 

1.  Symptoms  and  lesions  produced  in  animals  susceptible 

to  infection. 

(a)  Guinea-pigs. 

The  guinea-pig  is  the  most  suitable  animal  for  the  study  of  experimental 
diphtheria.  The  organism  may  be  introduced  either  under  the  skin,  into 
the  peritoneal  cavity,  into  the  trachea  or  on  to  mucous  surfaces. 

1.  Sub-cutaneous  inoculation. — 0'5  c.c.  of  a  twenty-four-hour  broth  culture 
inoculated  sub-cutaneously  will  kill  the  animal  in  24r-72  hours  according  to 
the  virulence  of  the  organism.     Following  the  inoculation  there  is  a  slight 
oedema  at  the  site  of  inoculation  and  a  rise  of  temperature  ;  the  animal  shows 
symptoms  of  illness  and  finally  dies. 

If  only  a  slightly  virulent  culture  be  used,  the  animal  may  recover  ;  in 
that  case  there  is  some  oedema  at  the  site  of  inoculation,  followed  by  a  slough 
which  heals  in  course  of  time.  [Similar  effects  result  from  the  inoculation  of 
sub-lethal  doses  of  fully  virulent  cultures.]  In  the  oedema  at  the  site  of 
inoculation  the  bacilli  multiply  for  the  first  6  or  8  hours  following  the  inocula- 
tion, after  which  multiplication  ceases  and  their  numbers  decrease  so  that 
post  mortem  relatively  few  organisms  are  found  in  the  clear  oedematous  fluid. 

The  organisms  do  not  pass  into  the  blood  and  internal  organs.  Post 
mortem,  there  is  a  very  marked  congestion  of  the  internal  organs  and  especially 
of  the  supra-renal  capsules,  and  a  large  pleural  effusion.  The  fluid  is  occa- 
sionally blood-stained  :  it  contains  no  bacilli. 

2.  Intra-peritoneal  inoculation. — The  symptoms  following  intra-peritoneal 
inoculation  are  less  severe  than  after  sub-cutaneous  inoculation  :    death  is 
longer  delayed  and  does  not  occur  till  between  the  fourth  and  the  twelfth 
day.     Over  and  above  the  ordinary  visceral  lesions  there  is  an  effusion  of 
fluid  into  the  peritoneal  cavity  and  it  is  only  in  this  situation  that  bacilli 
can  be  found. 

3.  Intra-tracheal    inoculation. — Tracheotomy    is    first    performed ;     the 
tracheal  mucous  membrane  is  then  abraded  and  a  portion  of  a  culture  of 
diphtheria  bacilli  applied.     A  false  membrane  forms  on  the  abraded  surface 
which  sets  up  a  true  condition  of  croup  rapidly  followed  by  death.     An 
essential  condition  of  success  is  that  the  mucous  membrane  be  traumatized  ; 
the  bacilli  fail  to  develop  on  the  uninjured  membrane. 


248  THE   DIPHTHERIA   BACILLUS 

4.  Infection  of  mucous  membranes. — False  membranes  may  be  produced 
by  applying  traces  of  culture  to  the  scarified  surfaces  of  the  conjunctiva  or 
vulva  of  the  guinea-pig. 

(b)  Rabbits. 

The  rabbit  is  far  less  susceptible  to  the  diphtheria  bacillus  than  the 
guinea-pig  and  only  succumbs  to  the  inoculation  of  very  virulent  cultures. 

1.  Sub-cutaneous  inoculation. — The  inoculation  of  2  c.c.  of  a  very  virulent 
broth  culture  leads  to  the  death  of  the  animal  in  about  5  days.     There  is 
an  oedema  at  the  site  of  inoculation  :    the  internal  organs  are  congested  and 
dotted   with   hremorrhagic   points  :     the   inguinal   and   axillary   glands   are 
swollen,  the  liver  jaundiced  and  friable  and  shows  fatty  degeneration  :  as  a  rule 
the  lungs  are  normal :   rarely  there  is  some  effusion  into  the  pleural  cavities. 
A  sub-lethal  dose  leads  to  paralysis,  affecting  chiefly  the  hind-quarters. 

2.  Intra-peritoneal  inoculation. — The  results  of  intra-peritoneal  inoculation 
are  less  severe,  and  death  only  takes  place  after  some  lapse  of  time.     The 
lesions  are  similar  to  those  mentioned  above. 

3.  Intra-venous  inoculation. — Following  the  inoculation  of  1-2  c.c.  of  a 
virulent  culture  into  an  ear  vein  death  takes  place  in  from  30-60  hours.     At 
the  post-mortem  examination  an  acute  nephritis  in  addition  to  the  ordinary 
lesions  is  found  :    the  organisms  do  not  multiply  in  the  blood  stream  being 
rapidly  taken  up  by  the  phagocytes  (Metin). 

4.  Inoculation  on  a  cutaneous  surface. — Roux  and  Yersin  obtained  excellent 
examples  of  false  membranes  by  blistering  a  small  area  on  the  internal  surface 
of  the  ear  and  then  applying  a  trace  of  a  culture  of  the  diphtheria  bacillus 
to  the   exposed  dermis.     It  is  essential   that   the   infected  surface  should 
not  be  allowed  to  dry  ;   the  ear  may  be  enclosed  in  a  small  rubber  bag.  care 
being  taken  that  the  vessels  are  not  compressed  at  the  base.     To  stop  the 
development  of  the  membrane  it  is  only  necessary  to  uncover  the  ear. 

5.  Intra-tracheal  inoculation. — A  typical  condition  of  croup  is  produced 
as  in  the  guinea-pig  but  more  easily. 

6.  Inoculation  on  mucous  surfaces. — The  results  are  the  same  as  in  the 
guinea-pig. 

(c)  Dogs. 

The  dog  is  susceptible  to  infection  with  the  diphtheria  bacillus. 

1.  Sub-cutaneous  inoculation. — Death  ensues  in  3  or  4  days.     Roux  and 
Yersin  noted  oedema  at  the  site  of  inoculation,  jaundice  and  finally  a  pro- 
gressive paralysis  :    the  fluid  of  the  oedema  contained  bacilli  but  the  blood 
was  sterile. 

2.  Intra-tracheal  inoculation. — Roux  and  Yersin  produced  a  swelling  of 
the  neck,  jaundice,  complete  paralysis,  and  death  on  the  fourth  day.     Post 
mortem  no  false  membrane  was  found  in  the  trachea. 

(d)  Cats. 

Death  follows  sub-cutaneous  inoculation  in  6-13  days.  A  cat  fed  on  milk 
from  a  cow  [said  to  be]  suffering  from  diphtheria  with  ulcers  on  the  udder 
also  contracted  the  disease  (Klein). 

(e)  Cows. 

Klein  [claims  to  have]  shown  that  the  cow  can  contract  diphtheria  both  by 
spontaneous  infection  and  as  a  result  of  experimental  inoculation  (vide  ante). 

Cows  inoculated  with  a  young  and  virulent  culture  of  the  diphtheria  bacillus 
died  with  congestion  of  the  internal  organs  ;  but  using  agar  cultures  several  days 
old,  Klein  was  unable  to  set  up  a  fatal  disease.  This  observer  on  several  occasions 
noted  an  eruption  on  the  udder  [of  cows  which  had  not  been  experimentally  infected] 


MIXED   INFECTIONS  249 

which  commenced  as  a  papule,  became  a  vesicle,  then  a  true  pustule  and  finally  an 
ulcer :  the  diphtheria  bacillus  was  found  in  the  vesicles  and  was  traced  on  many 
occasions  into  the  milk.  Klein  observed  a  similar  eruption  on  two  cows  which 
succumbed  to  the  inoculation  of  a  very  virulent  culture.  [Other  observers  have 
failed  to  confirm  these  experiments.] 

(/)  Birds. 

Pigeons  and  fowls  rapidly  succumb  to  the  inoculation  of  a  broth  culture 
of  the  diphtheria  bacillus  injected  sub-cutaneously  or  into  the  pectoral 
muscles  in  doses  of  1  c.c.  :  death  takes  place  in  less  than  60  hours. 
With  doses  of  less  than  O2  c.c.  the  animal  usually  recovers  ;  sometimes 
paralysis  is  observed.  Post  mortem  a  thin  greyish  film  and  a  gelatinous 
oedema  is  found  around  the  site  of  inoculation.  When  the  culture  has  been 
inoculated  into  the  muscle  the  latter  is  swollen  and  its  fibres  have  an  ochre 
tint :  the  internal  organs  are  intensely  congested. 

Following  inoculation  of  the  bacillus  into  the  larynx  these  animals  suffer 
from  croup  as  do  rabbits. 

Small  birds  (sparrows,  chaffinches,  etc.)  are  highly  susceptible  and  rapidly 
succumb  to  sub-cutaneous  inoculation. 

(g)  Rats  and  mice  are  immune  to  diphtheria. 

To  sum  up,  it  is  characteristic  of  the  diphtheria  bacillus  that  it  cannot  pene- 
trate the  tissues  of  susceptible  animals  ;  it  remains  localized  at  the  site  of  inocula- 
tion and  even  in  this  situation  its  development  is  quickly  arrested,  so  that  passage 
through  a  series  of  animals  rapidly  becomes  impossible. 

2.  Influence  of  other  organisms  on  the  clinical  course 
of  the  disease. 

Roux  and  Yersin  have  shown  that  certain  other  organisms  may  be  associated 
with  the  diphtheria  bacillus  and  at  times  play  an  important  part  in  the  clinical 
manifestations  of  the  disease.  The  diphtheria  bacillus  is  rarely  found  in 
pure  culture  in  the  false  membranes  :  occasionally  the  organisms  associated 
with  it  are  few  in  number  and  of  no  clinical  importance,  but  it  often  happens 
that  a  considerable  number  of  other  bacteria  are  present  many  of  which  play 
an  important  role  in  the  clinical  course  of  the  disease  and  to  some  extent 
determine  its  severity. 

Martin  has  pointed  out  that  the  mere  presence  of  a  few  other  organisms  with  the 
diphtheria  bacillus  in  cultures  sown  with  the  material  from  a  case  of  diphtheria 
is  not  sufficient  evidence  upon  which  to  base  a  diagnosis  of  secondary  infection  ; 
such  an  infection  can  only  be  diagnosed  when  the  number  of  other  organisms  is  very 
considerable.  And  it  is  in  this  connexion  that  a  preliminary  microscopical  examina- 
tion of  the  material  from  the  throat  is  of  great  importance,  because  it  can  then  be 
determined  whether  any  associated  bacillus  has  multiplied  therein  and  whether  it  is 
or  is  not  the  predominant  organism  present :  whereas  when  the  material  is  sown  on 
culture  media  one  species  of  organism  (perhaps  sparsely  represented  originally)  may 
outgrow  all  others,  and  moreover  the  presence  of  anaerobic  organisms  may  pass 
entirely  unnoticed.  It  must  be  remembered  that  the  surface  of  the  membrane 
may  be  contaminated  by  the  different  organisms  of  the  mouth,  and  must  therefore 
be  cleansed  before  material  is  removed  for  investigation. 

The  following  are  the  most  important  of  the  organisms  which  may  be 
found  in  association  with  the  diphtheria  bacillus  : — 

(i)  Brisou's  coccus. — Roux  and  Yersin  and  also  Martin  drew  attention  to 
a  small  coccus  which  they  frequently  found  associated  with  the  diphtheria 
bacillus  ;  they  called  it  the  Brisou  coccus  after  the  name  of  the  child  from 
whom  they  first  isolated  it.  The  organism  occurs  either  in  the  form  of  single 
cocci  or  as  diplococci  or  in  clusters.  It  is  gram-positive.  On  coagulated 


250  THE   DIPHTHERIA   BACILLUS 

serum  the  colonies  are  small,  whitish  in  colour  and  almost  transparent, 
slightly  raised  and  circular.  It  is  not  pathogenic  to  laboratory  animals. 
Usually,  though  not  always,  the  association  of  this  organism  with  the 
diphtheria  bacillus  is  unimportant  from  the  point  of  view  of  prognosis. 

(ii)  Staphylococcus  pyogenes. — Staphylococci  constitute  a  more  serious 
complication  than  the  preceding  :  respiratory  complications  are  frequent. 
In  a  case  in  which  the  staphylococcus  aureus  was  associated  with  the  diphtheria 
bacillus  the  author  observed  a  considerable  swelling  of  the  neck  during 
convalescence. 

(iii)  Streptococci. — According  to  Martin  a  secondary  infection  with  strepto- 
cocci produces  the  most  severe  form  of  diphtheria  ;  broncho-pneumonia 
frequently  supervenes  in  cases  where  streptococci  are  found  with  the  diphtheria 
bacillus. 

Metin  demonstrated  by  experiment  upon  guinea-pigs  the  unfavourable  influence 
of  Staphylococci  and  streptococci  upon  the  course  of  the  infection  :  he  found  that 
the  diphtheria  bacillus  in  such  cases  multiplied  in  the  blood  stream  and  in  the  internal 
organs  and  was  present  in  enormous  numbers  at  the  site  of  inoculation. 

(iv)  Bacillus  coli. — -The  colon  bacillus  is  not  infrequently  found  in  the 
mouths  of  healthy  persons,  and  it  is  therefore  to  be  expected  that  it  should 
be  found  in  the  false  membranes  in  some  cases  of  diphtheria.  The  multipli- 
cation of  this  organism  is  a  serious  complication  ;  three  cases  recorded  by 
Blasi  and  Russo-Travalli  terminated  fatally.  These  observers  grew  the 
diphtheria  bacillus  and  the  colon  bacillus  together  and  showed  that  the 
toxicity  of  diphtheria  cultures  was  increased  considerably. 

(v)  Other  organisms. — Association  with  the  pneumococcus,  with  the 
pneumobacillus  of  Friedlander,  with  the  proteus  vulgafis,  Vincent's  bacillus 
fusiformis  and  the  anaerobic  organisms  of  the  mouth  (Chap.  XXXIX.)  has 
also  been  recorded. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  diphtheria  bacillus  is  a  highly  pleormorphic  organism  ;  it  is  non- 
motile,  and  does  not  form  spores.  [In  general  terms  it  may  be  described  as 
a  small,  slender,  straight  or  slightly  curved,  usually  irregularly-staining  rod 
with  rounded  and  sometimes  swollen  ends  :  curved  bacilli  with  swollen  ends, 
resembling  a  gherkin  in  appearance,  are  very  characteristic.  In  size  it  is  sub- 
ject to  considerable  variation.] 

Various  attempts  have  been  made  to  classify  the  different  varieties  of 
diphtheria  bacilli.  Three  varieties  may  be  distinguished  depending  upon 
the  length  of  the  organism,  viz. 

(a)  Short  bacilli  almost  like  cocci.     They  measure  about  2/j.  x  0'8/x  and  often 
occur  in  pairs  arranged  parallel  to  one  another  (p.  254). 

(b)  Bacilli  of  intermediate  size,  measuring  3-4/^t  x  O8/*.     These  bacilli  are 
arranged  parallel  to  one  another  or  are  found  in  pairs  end  to  end,  the  latter 
often  forming  an  acute  angle  like  the  letter  V  or  a  circumflex  accent. 

(c)  Long  bacilli  4-5/x  or  more  in  length.     In  culture  they  are  seen  to  be 
interlaced  and  without  definite  arrangement,  very  like  brushwood.     These 
bacilli  generate  the  most  potent  samples  of  toxin  (p.  257)  and  are  usually 
found  in  severe  cases  of  diphtheria.     On  the  other  hand,  the  short  bacilli 
(group  a)  are  as  a  rule  almost  avirulent  (Martin). 

[Another  basis  of  classification  of  diphtheria  bacilli  is  that  worked  out  by 
Cobbett.  This  observer  relied  on  the  staining  reactions  alone  and  was  thus 
able  to  distinguish  five  groups  of  diphtheria  bacilli  in  young  serum  cultures. 


MORPHOLOGY 


251 


(Size  and  form  were  as  will  be  seen  found  to  be  in  close  relation  to  similarity 
in  staining  reaction.) 

(a)  Irregularly  beaded    bacilli — long  and  faintly  stained — -the  type  most 
frequently  seen. 


.  FIG.  165.  FIG.  166. 

FIGS.  163,  164,  165,  and  166. — Types  of  diphtheria  bacilli  from  young  serum  cultures. 

Mounted  in  dilute  Lceffler's  blue  (1  in  5  with  water).     Oc.  4  ;  obj.  yVth,  Zeiss. 

(/3)  Regularly  beaded  bacilli — streptococcal  forms — stain  darkly  and  may 
be  mistaken  for  streptococci. 

(y)  Barred,  segmented  or  banded  forms. 

(6)  Uniformly  stained  bacilli. 

(e)  Oval  bacilli  with  one  unstained  septum. 

[This  last  type  is  found  in  greatest  numbers  in  very  young  cultures  :  they 
are  probably  young  forms,  and  it  is  to  be  noted  that  individuals  of  this  type 
are — morphologically — -practically  indistinguishable  from  typical  forms  of 
Hofmann's  bacillus.] 

Branched  forms  of  the  diphtheria  bacillus  have  been  described  by  Babes, 
Escherich,  Concetti  and  others. 

Involution  forms  in  which  one  or  both  ends  are  swollen  giving  the  organism 
a  pear-shaped,  clubbed,  or  dumb-bell-like  appearance  are  sometimes  met 
with  in  old  cultures  and  in  smears  made  from  the  false  membrane. 

Staining  methods. 

(a)  The  diphtheria  bacillus  stains  readily  with  the  basic  aniline  dyes. 
Films  from  cultures  or  membranes  may  be  stained  with  diluted  Loeffler's 


252  THE  DIPHTHERIA   BACILLUS 

alkaline  methylene  blue  [1  to  4  of  water],  Roux's  blue  or  with  dilute  Ziehl's 
fuchsin.  [The  first  of  these  is  the  stain  strongly  recommended.] 

[The  method  recommended  is  that  devised  by  Cobbett.  Spread  films, 
dry  and  fix  in  the  ordinary  manner.  Wash  in  10  per  cent,  acetic  acid.  Wash 
in  water.  Mount  in  a  drop  of  Lceffier's  blue  diluted  5  times  with  water.  Blot. 
Examine.] 

When  stained  with  methylene  blue  [and  especially  with  a  dilute  blue] 
the  bacilli  are  found  to  be  irregularly  stained,  granules  being  seen  which  stain 
more  deeply  than  the  protoplasm  :  these  granules  or  metachromatic  bodies 
of  Babes  are  sometimes  called  polar  bodies.  German  writers,  in  the  deter- 
mination of  the  diphtheria  bacillus,  attach  great  importance  to  their  presence. 
[But  though  the  majority  of  diphtheria  bacilli  show  these  Babes'  bodies, 
some  types  stain  uniformly ;  moreover  some  bacilli  other  than  diphtheria 
bacilli  also  show  deeply-staining  granules.]  The  diphtheria  bacillus,  especi- 
ally in  old  cultures,  frequently  shows  irregular  vacuolated  spaces  which  do 
not  stain,  whatever  dye  be  used ;  it  is  to  be  noted  that  these  are  not  spores. 

(b)  Gram's  method. — The  diphtheria  bacillus  is  gram-positive.     Decolour- 
ization  must  not  be  pushed  too  far  because  the  organism  will  not  resist  a 
prolonged  action  of  alcohol.     Gram's  method  gives  beautiful  preparations 
with  smears  and  sections  of  false  membranes. 

(c)  Special  methods,     (a)  Neisser's  stain. — To  bring  out  the  polar  bodies  the 
method  of  Ernst-Neisser  may  be  applied. 

Two  solutions  are  necessary — 

A.  Methylene  blue  (Griibler),  -  1  gram. 
96  per  cent.  Alcohol,  -          -  20  c.c. 
Distilled  water,  -  950     „ 
Glacial  acetic  acid,    -  -         -  50     ,, 

Dissolve  the  blue  in  the  alcohol,  then  add  the  water  and  acid. 

B.  Vesuvin,  -  0'5  gram. 
Boiling  distilled  water,       -                                                             -         250      c.c. 

A  cover-glass  preparation  is  made  with  a  drop  of  an  emulsion  of  an  eigh teen- 
hour-old  culture  on  serum.  This  is  left  in  the  acid  solution  of  blue  for  1-3 

hours,  washed  in  water,  stained  for  a  few  seconds 

':';•":.        r  •'  in    the    vesuvin    solution,    washed    again    and 

•'  •    .  '         mounted.     [The    method    may  be    modified  by 

V^  staining  for  1  minute  in  each  of  the  blue  and 

*^'     $'      ,-"      *•.'":.:'"    brown  solutions,  washing  in  water  between  the 

.£*£  two  operations.] 

.*.     :.v."        x«.'.         So  stained  the  diphtheria   bacillus  is  brown, 

\    v^  *     '/--•  with  deep  blue  [or  violet]  granules  situated  as 

f/.    '"*'**{       '    r  a  rule  at  the  poles  or  ends.     (The  pseudo-diph- 

,  rt         theria  [Hofmann]   bacillus,   on  the  other  hand, 

<T£.  according  to  Neisser,  never  shows  polar  bodies, 

but  is  stained  uniformly  brown  or  has  some  blue 

staged  bv'L^r'fmSd.  "Sf"  gjaif  ^regularly  distributed  through  the  body 
Obj.  Ath  Zeiss.  ot  the  organism.  It  is  now  admitted  that  this 

reaction  is  not  characteristic.) 

[(/?)  The  following  is  a  modification  of  Neisser's  stain  which  apparently 
gives  better  results  than  that  just  described. 
Prepare  two  solutions  : 

A.  Methylene  blue  (Grubler), 1  gram. 

Absolute  alcohol.       -         -         -         .         .         .         .         .  50  c  c 

Glacial  acetic  acid,    -         -         -         .         .         .         .         .  50 

Distilled  water,                    ----...  1000 


CULTURAL  CHARACTERISTICS  253 


B.  Crystal  violet  (Hochst),      -  1  gram. 

Absolute  alcohol,       -  10  c.c. 

Distilled  water,  -         300    „ 

1.  Stain  for  1  second  or  longer  in  a  mixture  made  just  before  use  of  2  parts 
of  A  and  1  part  of  B. 

2.  Wash  rapidly  in  water. 

3.  Counterstain  for  3  seconds  in  cresoidin  solution. 

Cresoidin,         -  1  part. 

Warm  water,    -  -         300  parts. 

When  dissolved,  filter. 

4.  Wash  in  water.  ] 

(7)  For  staining  the  polar  bodies  Epstein  recommends  pyronin.  Stain 
for  20  seconds  in  pyronin',  wash,  treat  with  Gram's  solution  for  10  minutes, 
dry  and  mount.  The  polar  bodies  are  stained  bright  red,  the  bacilli  pale  red. 

Note. — The  diphtheria  bacillus  becomes  decolourized  very  quickly  when  mounted 
in  balsam.  For  permanent  preparations  the  following  method  is  recommended. 

1.  Stain  in  dilute  Ziehl's  fuchsin  for  1—3  minutes.     Wash  in  water. 

2.  Stain  in  the  same  manner  with  Roux's  blue.     Wash,  dry,  and  mount. 

2.  Cultural  characteristics. 

A.  Conditions  of  growth. — The  diphtheria  bacillus  will  grow  at  all  tempera- 
tures between  20°  C.  and  40°  C.  but  not  above  42°  C.,  the  optimum  being 
35°-37°  C.     The  bacillus  is  aerobic  :   some  growth  however  takes  place  under 
anaerobic  conditions,  but  it  is  poor  and  the  organism  rapidly  loses  its  vitality. 

B.  Media.     1.  Broth. — Peptonized- veal-broth  gives  a  /better  growth  than 
beef-broth. 

After  incubating  at  37°  C.  for  12-24  hours  small  white  masses  of  growth 
will  be  seen  adhering  to  the  sides  of  the  flask  and  later  a  film  forms  on  the 
surface  :  if  examined  microscopically  this  film  will  be  found  to  consist  of 
tangled  masses  of  bacilli  many  of  the  latter  being  club-shaped.  Finally, 
a  deposit  forms  at  the  bottom  of  the  tube  leaving  the  supernatant  fluid 
clear.  The  best  way  to  obtain  rapidly  a  luxuriant  growth  is  to  sow  the 
bacillus  in  a  flat  flask  and  pass  a  current  of  air  over  the  growth  during 
incubation. 

For  this  purpose  select  a  flask  similar  to  that  (Fern- 
bach's)  shown  in  fig.  168.  Fill  it  with  broth  through 
the  central  vertical  tubulure  until  the  level  of  the  fluid 
reaches  to  just  below  the  openings  of  the  lateral  tubu- 
lures  :  plug  all  three  openings  with  wool,  sterilize  in 
the  autoclave,  sow  through  the  vertical  tubulure, 
replace  the  wool  plug  and  cover  it  with  an  india-rubber 
cap.  Now  attach  one  of  the  lateral  tubes  to  a  water 
pump  and  draw  a  slow  current  of  air  through  the  flask ; 
the  air  entering  by  the  other  tube  sweeps  in  a  con- 
tinuous  stream  over  the  surface  of  the  broth. 

When  the  diphtheria  bacillus  is  grown  in  broth  the  reaction  of  the  latter 
first  becomes  acid,  but  after  a  few  days  this  initial  acidity  is  converted  into 
an  alkaline  reaction  accompanied  by  a  precipitate  of  ammonio-magnesium 
phosphate.  In  media  containing  glycerin  the  acid  reaction  is  very  marked 
and  persistent,  and  the  bacilli  rapidly  die  in  such  media. 

Martin's  broth  (p.  33)  is  better  than  ordinary  broth  and  in  it  growth  is 
very  luxuriant,  the  medium  never  becomes  acid  and  the  virulence  of  the 
bacilli  is  maintained  for  a  long  time. 

2.  Coagulated  serum. — On  coagulated  serum  which  is  the  best  medium  for 
the  diphtheria  bacillus  growth  is  very  rapid. 


254 


THE   DIPHTHERIA   BACILLUS 


(a)  Isolated  colonies  (grown  on  the  surface). — After  18  hours  (at  37°  C.) 
greyish  white  points  are  seen  which  rapidly  grow  to  the  size  of  a  pin's  head  : 
by  transmitted  light  the  colonies  are  more  opaque  in  their  centres  than  at 

their  margins  :  as  growth  proceeds  they 
attain  a  diameter  of  5  mm.,  remain  regular 
and  are  sometimes  pale  yellow  in  colour. 

(ft)  Stroke  cultures. — Colonies  similar  to 
those  described  above  appear  along  the  line 
of  sowing  ;  these  soon  become  confluent  and 
form  a  fairly  broad  greyish  band  with  ir- 
regularly serrated  margins. 

The  long,  short  and  medium  sized  varieties  of 
the  diphtheria  bacillus  cannot  be  distinguished 
by  the  characters  of  the  growth  on  serum  ; 
colonies  of  the  short  variety  are,  however,  some- 
times whiter  and  moister  than  usual ;  these 
whiter  colonies  will  be  found  to  grow  at  room 
temperature,  and  are  in  fact  colonies  of  the 
pseudo-diphtheria  or  Hofmann's  bacillus  (vide 
infra). 

3.  Agar. — Colonies  on  agar  are  very  similar 
to  those  on  serum  though  sometimes  larger 
and  whiter.     Growth  is  somewhat  slower. 

4.  Gelatin. — Stab  cultures  in  gelatin  (15 
per  cent.)  at  22°-240  C.  give  rise  to  a  very 
poor  growth  consisting  of  small  white  punc- 
tiform  colonies  along  the  line  of  the  stab. 

us169  -Cultures  of  the  diPhtheria  The    diphtheria   bacillus   does   not    liquefy 

1.  Surface  culture  on  agar  (3  days   at    gelatin. 

Ill  hours)2'  IS°lated  COl°nieS  °n  Serum       5-  Potato.— The  diphtheria  bacillus   does 

not  appear  to  grow  on  potato  ;  some  ob- 
servers have  however  described  a  delicate  growth  consisting  of  an  almost 
invisible  yellowish  glaze. 

6.  Egg  albumin. — SakarofE  recommends  white  of  egg  coagulated  by  heat 
as  a  medium  in  place  of  blood-serum  (p.  .53,  B).     When  sown  on  the  surface 
of  this  medium,  and  incubated  for  24  hours,  the  diphtheria  bacillus  grows  in 
the  form  of  small,  dull,  slightly  transparent,  hemispherical  colonies  somewhat 
darker  in  colour  than  the  medium  :    sometimes  towards  the  twelfth  day  the 
growth  may  be  of  a  brown  or  flesh  colour. 

7.  Milk. — The  diphtheria  bacillus  grows  luxuriantly  in  fresh  milk.     The 
medium  is  not  coagulated. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Vitality  and  virulence. 

Vitality. 

The  diphtheria  bacillus  remains  alive  in  culture  for  a  considerable  length 
of  time :  sub-cultures  can  be  grown  from  a  culture  5  or  6  months  old. 

In  moist  cultures  the  bacillus  is  readily  destroyed  :  exposure  to  a  tem- 
perature of  58°  C.  for  a  few  minutes  is  sufficient  to  sterilize  a  broth  culture. 

When  dried,  the  bacilli  are  more  resistant  to  the  action  of  heat  and  can 
be  subjected  to  a  temperature  of  95°  C.  for  several  minutes  without  being 
killed. 

The  diphtheria  bacillus  shows  even  greater  powers  of  resistance  to  heat 


in 


VIRULENCE  255 


in  the  false  membranes  :  thus  a  false  membrane  may  be  dried  and  exposed 
to  a  temperature  of  95°-100°  C.  for  an  hour,  and  yet  the  bacilli  may  be  found 
to  have  escaped  destruction  when  cultures  are  sown. 

Drying  has  but  little  effect  on  the  vitality  of  the  diphtheria  bacillus  if 
albuminous  matter  be  present :  thus  Roux  and  Yersin  dried  a  piece  of  mem- 
brane and  kept  it  away  from  the  light  at  room  temperature  ;  portions  of 
it  sown  3  months  or  5  months  later  yielded  cultures  of  the  diphtheria  bacillus. 
Bacilli  from  serum  cultures  are  more  easily  destroyed  by  drying  especially 
if  they  be  rapidly  dried.  Light  has  considerable  bactericidal  properties  : 
thus  if  a  piece  of  false  membrane  be  dried  and  then  exposed  to  air  and  sun- 
light the  organisms  are  destroyed  in  a  relatively  short  space  of  time.  Roux 
and  Yersin,  for  instance,  found  no  living  bacilli  in  a  piece  of  membrane  which 
had  been  dried  and  then  exposed  to  air  and  sunlight  for  a  period  of  6  weeks. 
A  culture  on  serum  which  had  been  dried  and  spread  in  a  thin  layer  was  found 
to  be  sterile  after  an  exposure  of  24  hours  to  diffused  light  (Ledoux-Lebard). 

Antiseptics  rapidly  sterilize  cultures  of  the  diphtheria  bacillus  :  1  per  cent, 
carbolic  acid,  2  per  cent,  bichromate  of  potassium  etc.  kill  cultures  instantly. 

If  silk  threads  be  dipped  in  a  culture  of  the  diphtheria  bacillus  and  dried, 
it  will  be  found  that  the  organisms  on  the  dried  threads  are  more  resistant 
to  the  action  of  antiseptics  and  can  withstand  the  action  of  1  per  cent,  carbolic 
acid,  5  per  cent,  salicylic  acid  in  alcohol,  etc.  for  several  minutes  (Chantemesse 
and  Widal).  The  resistance  of  the  bacilli  in  false  membranes  to  antiseptics 
is  even  greater. 

Virulence. 

The  virulence  of  a  given  diphtheria  bacillus  must  be  tested  as  follows  : 

Sow  the  organism  in  broth,  incubate  at  37°  C.  for  24  hours,  inoculate  1  c.c. 
beneath  the  skin  of  a  guinea-pig  weighing  400-500  grams.  One  of  several 
results  may  follow  : 

(a)  In  the  case  of  a  very  virulent  bacillus  death  will  occur  in  24^30  hours. 

(/3)  In  the  case  of  a  bacillus  of  intermediate  virulence  the  animal  will 
succumb  in  2-6  days. 

(y)  In  the  case  of  a  slightly  virulent  organism  death  may  not  take  place 
for  8-10  days. 

(5)  In  the  case  of  a  bacillus  of  very  low  virulence  the  animal  will  survive 
but  a  local  oedema  followed  by  a  slough  will  form  at  the  site  of  inoculation. 

(e)  Finally,  should  the  bacillus  be  avirulent  no  lesion  whatever  will  follow 
the  inoculation. 

[It  will  be  necessary,  as  pointed  out  above,  to  make  certain  that  the 
bacillus  used  for  inoculation  is  growing  well  in  broth.] 

The  virulence  of  the  organisms  isolated  from  false  membranes  is  very 
inconstant :  in  severe  cases,  virulent  bacilli  are  very  numerous  :  in  mild  cases 
in  addition  to  colonies  of  virulent  bacilli,  numerous  colonies  of  non-virulent 
organisms  will  be  found.1 

[The  pathogenicity  of  different  strains  of  the  diphtheria  bacillus  when  first 
isolated,  as  tested  with  two-day  broth  cultures,  varies  greatly  (minimal 
lethal  dose  varies  as  400  to  1)  ;  and  the  virulence  of  washed  bacilli  from 
two-day  broth  cultures  of  different  strains  varies  at  least  as  much  as  the 
pathogenicity  of  whole  cultures  (Arkwright).] 

(a)  Attenuation  of  virulence. — In  old  cultures  the  diphtheria  bacillus  loses 
much  of  its  virulence  but  the  latter  can  be  fully  recovered  by  sowing  the 

[l  It  is  not  justifiable  to  assume  that  because  a  diphtheria  bacillus  is  non-pathogenic 
to  laboratory  animals  that  it  is  therefore  non -pathogenic  to  man  :  on  the  other  hand 
there,  is  some  evidence  to  show  that  "  non- virulent "  diphtheria  bacilli  are  capable  of 
producing  diphtheria  in  a  susceptible  human  subject.] 


256  THE   DIPHTHERIA   BACILLUS 

organism  in  broth.  On  the  other  hand,  when  the  organism  is  grown  at  39°  C. 
on  glycerin-agar  or  on  broth  with  a  current  of  air  passing  over  it,  it  loses  its 
virulence  rapidly,  so  that  on  inoculation  into  a  guinea-pig  nothing  more  than 
a  local  oedema  results  (Roux  and  Yersin). 

The  same  fact  may  be  observed  by  drying  a  false  membrane  from  a  case 
of  diphtheria  and  exposing  it  to  the  air  :  the  organism  remains  alive  for  a 
long  time,  but  if  fragments  of  the  membrane  be  sown  from  day  to  day  it 
will  be  found  that  the  number  of  non-virulent  colonies  increases.  The 
bacilli  thus  artificially  attenuated  have  all  the  characteristics  of  the  pseudo- 
diphtheria — Hofmann's— bacillus  (see  p.  273).  [It  would  seem  that  the 
explanation  of  this  result  is  to  be  found  in  the  supposition  that  both  the 
diphtheria  bacillus  and  Hofmann's  bacillus  were  present  in  the  original 
membrane  and  that  the  former  died  out.  All  attempts  to  convert  a  diph- 
theria bacillus  into  an  Hofmann  and  vice  versa  have  invariably  failed  (p.  274).  ] 

(b)  Restoration  of  virulence. — -It  is  impossible  to  restore  the  virulence  of 
an  organism  which  has  become  so  attenuated  as  to  have  entirely  lost  its  viru- 
lence for  the  guinea-pig  (Roux  and  Yersin). 

On  the  other  hand,  Roux  and  Yersin  succeeded  in  restoring  the  virulence 
of  an  organism  which  produced  nothing  more  than  a  slight  oedema  in  the 
guinea-pig  ;  they  inoculated  the  bacillus,  and  with  it  a  virulent  culture  of  a 
streptococcus,  into  a  guinea-pig  :  the  animal  succumbed  with  symptoms  of 
diphtheria  and  the  bacillus  recovered  from  the  fluid  of  the  local  oedema  was 
found  to  have  very  distinctly  increased  in  virulence.  According  to  Blasi 
and  Russo-Travalli  association  with  the  colon  bacillus  has  a  similar  effect 
in  restoring  the  virulence  of  the  diphtheria  bacillus.  Trumpp,  by  inoculating 
a  mixture  of  small  doses  of  diphtheria  toxin  and  an  almost  non-virulent 
bacillus,  was  also  able  to  restore  the  virulence  of  the  organism. 

(c)  Exaltation  of  virulence. — Roux  and  Yersin  failed  to  raise  the  virulence 
of  the  diphtheria  bacillus  by  passage  through  guinea-pigs  or  rabbits.     Bar- 
dach  after  passing  the  bacillus  through  twenty-five  dogs  noted  a  distinct 
increase  in  virulence  for  the  dog  but  only  a  slight  increase  in  virulence  for 
the  guinea-pig. 

Martin  grew  the  bacillus  in  collodion  sacs  in  the  peritoneal  cavities  of  a 
series  of  rabbits  and  succeeded  in  raising  the  virulence  for  that  animal,  but 
found  that  the  virulence  for  the  guinea-pig  was  unaltered. 

[Thus  the  virulence  of  the  diphtheria  bacillus,  while  easily  lowered,  is 
difficult  to  increase  by  laboratory  methods.] 

[2.  Bio-chemical  reactions.] 

[(a)  Action  on  carbohydrates. — "  All  strains  of  the  diphtheria  bacillus 
produce  acid  from  glucose,  galactose,  laevulose,  and  maltose.  Most  form 
acid  out  of  dextrine  and  glycerine.  On  lactose  the  action  is  very  variable, 
and  only  a  few  strains  act  on  saccharose.  All  tests  on  mannite  yielded 
negative  results  "  (Graham-Smith).  No  gas  is  formed  in  any  of  the  media. 

[In  testing  the  action  of  diphtheria  bacilli  upon  carbohydrates  the  most 
suitable  medium  to  which  to  add  the  carbohydrate  is  Hiss's  serum-water 
medium  (Chap.  XL.).  To  this  solution,  1  per  cent,  of  the  carbohydrate  is 
added. 

[If  sugar-free  broth  be  used  the  results  may  not  be  so  uniform,  because  as 
has  already  been  pointed  out  some  strains  of  the  diphtheria  bacillus  do  not 
grow  readily  on  such  broth  when  first  isolated  from  the  body. 

[(b)  Indol. — The  diphtheria  bacillus  does  not  appear  to  form  indol  (Theobald 
Smith  and  others),  but  some  observers  are  said  to  have  obtained  an  indol 
reaction  after  prolonged  cultivation.] 


P  DIPHTHERIA  TOXIN  257 

3.  Toxin. 
Hphtheria,  as  Roux  an,d  Yersin  showed,  is  an  intoxication  with  the  highly 
poisonous  products  of  the  diphtheria  bacillus.     These  products,  as  the  same 
observers  proved,  are  also  present  in  broth  cultures  of  the  bacillus. 

In  their  first  experiments  Roux  and  Yersin  were  only  able  to  manufacture 
a  very  weak  toxin  of  which  30  c.c.  were  required  to  kill  a  guinea-pig.  Martin 
now  prepares  a  toxin  which  is  fatal  to  adult  guinea-pigs  in  doses  of  ^J^  c.c. 
and  even  -^  c.c.  [Toxins  even  more  powerful  than  this  have  been  prepared.  ] 

(a)  Preparation  of  diphtheria  toxin. 

Conditions  under  which  toxin  is  elaborated. — Diphtheria  toxin  is  obtained 
by  growing  a  toxigenic1  bacillus  in  presence  of  air. 

A.  Selection  of  the  organism. — The  strain  to  be  used  for  the  preparation  of 
toxin  should  first  of  all  be  tested  on  animals.     To  be  suitable  for  the  purpose, 
1  c.c.  of  a  broth  culture  of  the  organism  should  when  inoculated  beneath  the 
skin  of  a  guinea-pig  weighing  300-400  grams  prove  fatal  in  24-36  hours. 
But  as  it  has  been  found  that  bacilli  isolated  from  very  severe  cases  of  diph- 
theria are  not  always  powerfully  toxigenic  [and  on  the  other  hand  some 
bacilli  are  toxigenic  and  non-virulent]  the  toxigenic  capacity  of  the  organism 
should  always  be  tested  before  embarking  upon  the  manufacture  of  large 
quantities  of  toxin.     [At  the  present  time  a  bacillus  known  as  Park  and 
Williams'  bacillus  No.  8  is  extensively  used  for  the  preparation  of  toxin. 
Park  and  Williams  recovered  this  organism  from  a  mild  tonsillar  case  of 
diphtheria.  ] 

To  preserve  the  toxigenic  properties  of  a  diphtheria  bacillus  sow  it  on  Martin's 
broth,  and  after  incubating  for  a  week  at  33°— 35°  C.  remove  the  tubes  from  the 
incubator  and  keep  them  in  the  dark.  Martin  keeps  48-hour  cultures  on  coagulated 
serum  at  10°-12°  C.  Old  cultures  stored  in  this  way  on  being  revived  by  a  couple 
of  sub-cultivations  yield  a  bacillus  which  has  a  very  considerable  power  of  toxin 
production. 

B.  The  choice  of  medium. — The  amount  of  toxin  produced  depends  upon 
the  composition  and  especially  upon  the  reaction  of  the  medium. 

It  has  already  been  pointed  out  that  when  the  diphtheria  bacillus  is  culti- 
vated upon  an  alkaline  broth  the  medium  is  first  turned  acid  but  after  a  few 
days  again  becomes  alkaline  ;  and  it  is  just  when  the  acid  reaction  begins  to 
diminish  that  the  toxin  begins  to  be  formed,  after  that  the  toxicity  of  the 
broth  increases  pari  passu  with  the  alkalinity  of  the  medium,  and  the  more 
rapidly  the  alkalinity  increases  the  more  rapidly  does  the  amount  of  toxin 
increase.  If  the  formation  of  acid  be  prevented  toxin  will  be  formed  both 
more  rapidly  and  also  in  larger  quantity.  As  the  result  of  experiment  many 
methods  have  been  devised  for  diminishing  or  altogether  preventing  the 
initial  formation  of  acid.  Roux,  Yersin  and  Martin  shortened  the  period 
of  acid  reaction  by  growing  in  a  current  of  air,  and  this  method  has  been 
used  for  a  long  time  in  the  preparation  of  toxin  on  a  large  scale.  Many 
observers  have  aimed  at  excluding  from  the  culture  medium  all  those  sub- 
stances (e.g.  glucose,  laevulose,  saccharose,  galactose,  glycerin)  from  which 
the  diphtheria  bacillus  forms  acid.  Nicolle,  for  instance,  obtained  a  satis- 
factory toxin  by  using  fresh  meat — meat,  that  is,  in  which  the  glycogen  had 
not  had  time  to  be  converted  into  glucose  (glycogen  not  being  convertible 
into  acid)  ;  Spronck  on  the  contrary  suggested  the  use  of  meat  which  was 

[l  The  disease-producing  power  of  a  diphtheria  bacillus  may  be  regarded  as  made  up 
of  two  elements,  toxicity  and  virulence.  The  former  represents  the  rate  of  accumulation 
by  it  of  toxin  in  culture  fluids,  the  latter  the  behaviour  of  the  bacillus  towards  living  tissue 
(Theobald  Smith).] 

R 


258 


THE   DIPHTHERIA   BACILLUS 


rotten  and  from  which  the  sugars  had  vanished.  Park  and  Williams  in  their 
investigations  used  a  broth  previously  made  alkaline  with  soda  :  Mace 
employed  a  broth  to  which  calcium  carbonate  had  been  added.  None  of 
these  methods  however  give  results  as  good  as  those  obtainable  by  the  method 
devised  by  Martin.  This  observer  succeeded  in  finding  a  medium  in  which 
no  acid  reaction  is  developed  and  which  yields  highly  toxic  cultures. 

1.  Method  of  Roux  and  Martin. — The  bacillus  is  grown  in  a  current  of  air, 
and  for  this  purpose  a  flask  (modified  from  Fernbach's)  similar  to  that  shown 
in  the  illustration  (fig.  170)  is  very  convenient. 


FIG.  170. — Flask  arranged  for  the  growth  of  the  diphtheria  bacillus  for  toxin 
production  (Roux  and  Martin's  method). 

Pour  into  each  flask  400-500  c.c.  of  veal  broth  ;  this  quantity  should  not  form 
a  layer  more  than  2  or  3  cm.  deep,  and  the  surface  of  the  broth  should  be  below  the 
opening  of  the  lateral  tube  D.  Plug  the  lateral  tube  D  and  the  neck  of  the  flask  C 
with  cotton- wool :  autoclave  the  flask  and  its  contents  :  allow  to  cool  and  sow  the 
medium  through  the  neck  C.  Incubate  at  37°  C.  and  after  about  24  hours  or  so — 
when  the  growth  is  well  started  and  the  broth  has  become  cloudy — arrange  the  flask 
so  that  a  current  of  air  can  be  passed  over  the  surface  of  the  broth,  thus  : — Into  the 
neck  of  the  flask  B  and  above  the  wool  plug  fit  an  india-rubber  plug  through  which 
a  piece  of  glass  tubing,  6,  bent  at  right  angles  is  passed,  and  connect  this  with  a 
second  flask  A  containing  a  little  water  through  which  the  air  is  made  to  bubble. 
Attach  the  tube  D  by  means  of  a  piece  of  india-rubber  tubing  to  a  water  pump. 
When  the  water  is  turned  on  air  is  bubbled  through  the  water  in  A — where  it  is  satu- 
rated with  moisture — and  drawn  over  the  surface  of  the  culture  in  B  which  is  thus 
aerated.  By  means  of  a  clip  on  the  tube  connecting  the  flasks  A  and  B  the  amount 
of  air  can  easily  be  regulated  so  that  a  constant 'but  not  violent  stream  of  air  can  be 
drawn  through  the  flask. 

After  incubating  for  3  or  4  weeks  the  culture  is  sufficiently  rich  in  toxin. 
At  the  bottom  of  the  flask  there  is  a  deposit  of  micro-organisms  and  on  the 
surface  a  thin  layer  of  young  bacilli.  The  reaction  is  strongly  alkaline. 

The  culture  is  now  filtered  through  a  Chamberland  bougie  by  one  of  the 
methods  already  described  (Chap.  I.).  The  filtrate  kills  adult  guinea-pigs 
in  doses  of  O'l  c.c. 

2.  Martin's  method.  Method  recommended. — The  need  for  the  current 
of  air  is  obviated,  and  hence  also  the  necessity  for  complicated  apparatus  : 
it  is  quicker  than  the  method  just  described  and  yields  moreover  a  more 
powerful  toxin.  A  dose  of  -*  J^th  c.c.  of  this  toxin  is  sufficient  to  kill  a  guinea- 

Pig- 

Martin  uses  a  peptonized  veal  broth  (p.  33)  sterilized  by  filtration1  and 

1  If  sterilized  at  120°  C.  this  medium  does  not  give  such  good  results.  It  is  better  to 
filter  or,  failing  nitration,  to  sterilize  on  three  successive  days  at  100°  C. 


PREPARATION  OF  THE   TOXIN 


259 


distributed  in  thin  layers  (3-4  cm.  deep)  in  large  flasks.     The  organism  soon 
becomes  accustomed  to  the  medium,  and  grows  well  in  it. 

Character  of  growth. — A  film  forms  on  the  surface  during  the  first  24  hours  and 
increases  both  in  area  and  in  thickness  during  the  next  24  hours  (reject  all  organisms 
which  do  not  form  a  film  about  the  end  of  the  third  day).  If  the  culture  is  growing 
well  the  film  breaks  up  and  falls  to  the  bottom  ;  a  new  film  then  forms  and  this  in 
turn  sinks  to  the  bottom  about  the  sixth  day,  after  which  no  further  film  is  formed. 
The  medium  is  never  acid  to  litmus,  but  on  the  other  hand  about  the  second  to  the 
fourth  day  it  is  alkaline  to  phenol-phthalein. 

The  culture  should  be  filtered  about  the  end  of  the  first  week  when  its 
toxicity  is  at  its  maximum ;  after  about  a  fortnight  the  toxin  content  begins 
to  diminish. 

3.  [G.  Dean's  method.    Recommended. — This  is  a  less  complicated  method 
than  Martin's  and  is  somewhat  similar  to  that  described  by  Park  and  Williams. 

[The  broth  is  prepared  with  "  silverside  "  of  beef  and  the  meat  may  be  used 
either  perfectly  fresh  or  after  hanging  for  7-12  days  at  a  temperature  of  8°  C.  Free 
the  beef  from  all  fat  and  fascia  and  then  mince  very  finely.  To  each  pound  (about 
500  grams)  of  beef  add  1  litre  of  fairly  alkaline  tap  water.  Put  the  meat  and  water 
into  an  enamelled  saucepan,  cover  with  the  lid,  and  allow  to  boil  quietly  for  £-2 
hours.  Filter  through  Swedish  filter  paper,  thoroughly  squeezing  out  all  the  juice 
from  the  beef.  To  the  filtrate  add  2  per  cent.  Witte's  peptone  and  0'5  per  cent, 
sodium  chloride.  Steam  at  100°  C.  for  1  hour.  Filter.  Make  the  filtrate  neutral 
to  litmus  and  then  add  7  c.c.  of  normal  soda  per  litre  while  still  hot.  Steam  again 
for  1  hour.  Filter.  Distribute  in  Erlenmeyer  flasks.  Steam  at  100°  C.  for  20  minutes 
and  then  allow  the  temperature  to  run  up  to  120°  C.  before  turning  out  the  gas. 

[The  toxicity  in  the  case  of  39  toxins  prepared  by  Dean  with  this  medium  and  the 
American  bacillus  (p.  257)  is  shown  in  the  following  table. 
1  killed  within  6  days  at 

8  f 

9 

7 
7 
1 

5  did  not  kill  at  ^iW         ] 

4.  Other  methods. — The  methods  of  Spronck,  Park  and  Williams,  Nicolle, 
and  Mace  which  are  in  use  in  some  laboratories  are  less  reliable  than  those 
given  above. 

Spronck's  first  method. — This  was  based  upon  the  absence  of  glucose  in  stale  meat. 

Prepare  2  per  cent,  peptone  broth  in  the  ordinary  way  but  with  meat  which  has 
been  hanging  for  some  days  until  it  has  acquired  a  slight  smell.  Make  alkaline  and 
then  add  0'5  per  cent,  salt  and  a  little  calcium  carbonate.  Distribute  in  quantities 
of  300-400  c.c.  in  half  litre  flasks.  Sterilize.  Sow  when  cool.  Incubate  at  37°  C. 
for  3-4  weeks.  Filter. 

Spronck's  later  method. — This  was  based  upon  a  possibly  beneficial  action  of 
yeast  in  promoting  the  production  of  toxin. 

Revive  the  bacillus  by  sowing  first  on  coagulated  blood  serum  and  then  on  peptone 
yeast  water  (p.  37).  Sow  from  the  latter  on  to  a  shallow  layer  of  peptone  yeast 
water  contained  in  a  large  flat  flask.  After  incubating  for  24  hours  the  growth  has 
formed  a  continuous  pellicle  on  the  surface  and  at  the  end  of  a  week  the  content  of 
toxin  is  at  its  maximum  :  a  dose  of  ^fa  c.c.  suffices  to  kill  a  guinea-pig.  Spronck 
does  not  use  a  porcelain  filter  but  adds  3  grams  of  carbolic  acid  per  litre  and  filters 
through  paper. 

Massol's  method. — Proceed  as  in  Spronck's  method  but  use  the  following  medium  : 

High  veal, 500  grams. 

Peptone  (Witte), 20      „ 

Water,   -  ...       1000      „ 

Neutralize.  Add  7  c.c.  normal  soda  solution.  Filter  through  filter  paper.  Steri- 
lize by  filtration  through  a  Chamberland  bougie. 


260  THE   DIPHTHERIA  BACILLUS 

Nicolle's  method. — Use  beef  killed  the  same  morning.  Mince  the  meat.  Add 
twice  its  weight  of  water  and  allow  to  stand  for  an  hour  at  10°  or  12°  C.  Add  2  per 
cent,  peptone  and  0*5  per  cent,  common  salt.  Heat  to  boiling  point.  Filter.  Make 
"  sufficiently  "  alkaline.  Heat  to  120°  C.  for  10  minutes.  Filter.  Distribute  in 
sterile  plugged  vessels.  Heat  to  115°  C.  for  15  minutes. 

Grown  in  this  medium  for  7  days  at  37°  C.  the  diphtheria  bacillus  will  yield,  with- 
out having  a  current  of  air  passed  over  the  culture,  a  toxin  quite  as  powerful  as 
that  obtainable  by  Roux  and  Martin's  method. 

Mack's  method. — Use  ordinary  peptone  broth  containing  in  addition  10  per  cent, 
calcium  carbonate.  Distribute  in  litre  or  two  litre  flasks  and  autoclave.  Incubate 
after  sowing  for  4-6  weeks  at  37°  C.  The  product  is  said  to  be  equally  as  toxic 
as  the  nitrate  prepared  by  Roux  and  Martin. 

Park  and  Williams'  method. — [vide  ante  Dean's  method.  ]  These  observers  grow 
the  bacillus  on  ordinary  peptone  broth  made  alkaline  to  the  extent  of  7  c.c.  normal 
soda  solution  per  litre  (p.  31). 

Protein-free  media. — Utchinsky  has  shown  that  it  is  possible  to  get  toxin  by 
growing  the  organism  on  media  containing  no  protein  and  consisting  merely  of 
salts  and  asparagin  (p.  39).  Hadley  prefers  to  substitute  glycocoll  (1  gram  per 
litre)  for  the  asparagin  in  Utchinsky's  medium. 

These  methods  always  yield  a  nitrate  very  weak  in  toxin.  Nicolle,  by  adding  to 
Utchinsky's  medium  peptone  Chapoteaut  and  gelatin  liquefied  by  B.  subtili-s,  gets 
a  very  powerful  toxin. 

(b)  The  testing  and  storing  of  toxin. 

(a)  The  testing  of  toxin. — The  toxin  content  of  the  product  manufactured 
with  the  same  bacillus  under  apparently  identical  conditions  is  subject  to 
considerable  variation  [vide  Dean's  results,  p.  259].  It  follows  therefore  that 
every  sample  of  toxin  must  be  tested. 

To  be  suitable  for  the  immunization  of  animals  (for  the  purpose  of  preparing 
a  therapeutic  serum)  a  toxin  must  kill  a  guinea-pig  weighing  400-500  grams 
in  48  hours  or  less  when  inoculated  in  quantities  of  0*1  c.c.  beneath  the 
skin. 

The  toxins  now  used  are  often  much  stronger  than  this  so  that  a  dose 
of  yj^  or  -0^(5-  c.c.  will  kill  a  guinea-pig  weighing  500  grams  in  36  hours. 

For  convenience  of  comparison  Ehrlich  has  suggested  the  adoption  of  a 
unit  of  toxin.  A  unit  of  toxin  is  the  quantity  necessary  to  kill  a  guinea-pig 
weighing  300  grams  in  96  hours  ;  and  a  toxin  is  said  to  contain  100,  200  etc. 
units  per  cubic  centimetre. 

For  measuring  small  quantities  of  toxin  it  is  convenient  to  make  dilutions  in 
sterile  water ;  for  example,  1  c.c.  of  toxin  added  to  9  c.c.  of  sterile  water  gives  a 
dilution  of  which  1  c.c.  represents  O'l  c.c.  of  toxin.  [Similarly  1  c.c.  of  No.  1  dilution 
added  to  9  c.c.  of  sterile  water  affords  a  dilution  of  which  1  c.c.  represents  O'Ol  c.c. 
of  toxin.  ] 

'  (ft)  To  store  toxin. — For  the  purpose  of  storing  toxin  use  sterile  [amber- 
coloured]  bottles,  which  must  be  exactly  filled,  well  plugged  and  kept  in  the 
dark.  Even  under  these  conditions  the  toxin  slowly  loses  its  toxic  properties. 

(c)  Action  of  toxin  on  animals. 

The  symptoms  which  follow  the  inoculation  of  diphtheria  toxin  into 
susceptible  animals  are  identical  with  those  produced  by  the  inoculation  of 
living  cultures  of  the  diphtheria  bacillus.  It  is  immaterial  whether  the  toxin 
be  administered  by  inoculation  beneath  the  skin,  into  the  peritoneal  cavity, 
into  the  veins  or  into  the  brain.  But  given  by  the  mouth  toxin  has  no 
effect  whatever. 

On  the  guinea-pig. — If  a  fraction  of  a  c.c.  of  toxin  (0' 1-0*25  c.c. — the  exact 
amount  depending  upon  the  toxin  content  of  the  filtrate)  be  inoculated 
beneath  the  skin  of  a  guinea-pig  an  oedema  rapidly  forms  at  the  site  of 


PROPERTIES   OF  THE   TOXIN  261 

inoculation ;  this  is  soon  followed  by  panting  respiration  and  death  super- 
venes in  20-30  hours.1 

Post  mortem  the  lesions  found  are  similar  to  those  described  as  following 
the  inoculation  of  living  bacilli. 

Smaller  doses  (0'005-0'002  c.c.)  of  a  powerful  toxin  kill  guinea-pigs  after 
the  lapse  of  5-30  days,  but  if  the  dose  be  too  small  the  animal  will  survive. 
Paralysis  is  very  rarely  seen  in  guinea-pigs. 

On  the  rabbit. — The  administration  of  0'25-0'5  c.c.  toxin  either  sub-cutane- 
ously  or  into  a  vein  terminates  in  death  accompanied  by  the  usual  lesions. 
If  the  dose  be  not  large  enough  to  kill  the  animal  very  rapidly  typical  diph- 
theria paralyses  develop. 

If  toxin  be  applied  to  mucous  membranes,  local  lesions  and  occasionally 
true  false  membranes  form  even  though  the  surface  be  intact  (Roger  and 
Bayeux,  Morax  and  Elmassian). 

On  the  dog. — Dogs  are  very  susceptible  to  the  action  of  diphtheria  toxin. 
A  dose  of  1  c.c.  sub-cutaneously  is  sufficient  to  kill  a  dog  rapidly  with  symptoms 
of  jaundice  and  diarrhoea  ;  lesions  will  be  found  in  the  liver  post  mortem. 
Smaller  doses  are  followed  by  paralyses  :  the  animal  may  recover  but  if 
death  takes  place  it  does  not  occur  so  rapidly  as  when  larger  doses  are 
used. 

On  birds. — Fowls,  pigeons  and  small  birds  rapidly  succumb  to  very  small 
doses  of  toxin  whether  the  inoculation  be  made  beneath  the  skin  or  into  the 
pectoral  muscle. 

Ruminants. — Goats  are  very  susceptible  to  diphtheria  toxin  ;  similarly, 
cows  often  succumb  to  the  inoculation  of  a  fraction  of  a  c.c.  of  toxin.  Sheep 
are  somewhat  less  susceptible, 

Horses. — The  horse  is  less  affected  by  diphtheria  toxin  than  ruminants ; 
but  a  dose  of  O'l  c.c.  of  a  toxin  of  which  the  lethal  dose  for  guinea-pigs 
was  75-iy  c.c.  has  been  known  to  kill  a  horse  weighing  400  kg.  The  ass  is 
more  susceptible. 

Rats  and  mice  are  nearly  immune  to  the  action  of  toxin  when  inoculated 
sub-cutaneously :  the  dose  of  toxin  required  to  kill  a  mouse  would  kill  as 
many  as  24  to  100  guinea-pigs  (Roux  and  Yersin). 

On  the  other  hand  intra-cerebral  inoculation  of  O'l  c.c.  of  toxin  kills  rats 
with  symptoms  of  diphtheria  paralysis  (Roux  and  Borrel). 

The  brain  of  the  rat  is  therefore  sensitive  to  the  action  of  diphtheria  toxin,  and 
the  reason  why  the  animal  does  not  die  as  the  result  of  sub-cutaneous  inoculation 
of  large  quantities  of  the  poison  is  because  the  latter  is  fixed  by  certain  cells  in  the 
tissues  (probably  by  the  phagocytes),  and  so  never  reaches  the  cerebrum. 

(d)  On  the  nature  and  properties  of  diphtheria  toxin. 

The  problem  of  the  nature  of  diphtheria  toxin  has  been  the  subject  of 
prolonged  and  extensive  investigations.  Brieger  and  Frsenkel  as  well  as 
Wassermann  and  Proskauer  regarded  toxin  as  a  tox-albumin,  and  Gamaleia 
considered  it  to  be  a  nucleo-albumin  :  but  these  observers  only  succeeded  in 
obtaining  very  impure  products  containing  relatively  very  little  toxin.  Roux 
and  Yersin  have  shown  that  the  active  principle  in  filtered  cultures  has  the 
chief  properties  of  enzymes.  A  temperature  of  100°  C.  destroys  diphtheria 
toxin  :  an  exposure  to  a  temperature  of  58°  C.  for  12  hours  lowers  its  toxicity 
to  such  an  extent  that  1  c.c.  of  the  heated  toxin  fails  to  kill  a  guinea-pig  ; 
and  the  effect  of  heating  to  70°  C.  is  to  attenuate  the  toxin  even  more.  In 
common  with  the  diastases,  diphtheria  toxin  has  the  property  of  being  carried 

[x  Sub-cutaneous  inoculation  is  always  followed  by  an  incubation  period  before  symptoms 
appear.] 


262  THE  DIPHTHERIA  BACILLUS 

down  in  the  precipitates  which  can  be  produced  in  the  solutions  in  which  it 
is  dissolved  (Miahle's  reaction). 

By  adding  a  solution  of  chloride  of  calcium  drop  by  drop  to  diphtheria  toxin 
phosphate  of  lime  is  precipitated  as  the  result  of  the  combination  of  the  calcium 
with  the  phosphates  present  in  the  liquid.  This  precipitate  when  collected  on  a 
filter  and  washed,  is  very  toxic  ;  the  sub-cutaneous  inoculation  of  a  mere  trace 
of  it  rapidly  causes  death  in  a  guinea-pig  accompanied  by  a  swelling  at  the  site  of 
inoculation  and  the  formation  of  a  small  greyish  false  membrane.  The  precipitate 
is  more  toxic  in  the  moist  than  in  dry  state.  Nevertheless  after  desiccation  it 
retains  most  of  its  toxic  properties,  and  in  this  condition  it  is  more  resistant  to  the 
action  of  heat  and  can  be  raised  to  a  temperature  of  70°  C.  without  losing  any  of 
its  toxicity  ;  and  further  a  very  small  amount  of  the  desiccated  precipitate  if  inserted 
beneath  the  skin  will  kill  three  guinea-pigs  in  succession  if  transferred  from  one 
animal  to  the  other  as  each  dies. 

After  filtering  off  the  first  precipitate  the  clear  filtrate  is  still  toxic,  and  precipi- 
tates may  be  produced  time  after  time ;  every  time  the  precipitate  contains  toxin 
but  in  a  progressively  diminishing  quantity,  until  finally  the  nitrate  will  no  longer 
produce  a  precipitate  though  it  is  still  slightly  toxic,  as  is  shown  by  the  fact  that 
when  inoculated  into  guinea-pigs  in  very  large  doses,  it  sets  up  a  chronic  intoxication. 

Diphtheria  toxin  is  soluble  in  water  but  again  like  the  diastases  is  precipi- 
tated by  alcohol :  but  precipitation  with  alcohol  diminishes  its  toxicity. 

To  precipitate  the  toxin  it  is  best  to  evaporate  the  filtrate  first  to  one-tenth  its 
volume  in  vacua  at  25°  C.,  and  then  to  add  -to  the  liquid  extract  4-5  volumes  of 
strong  alcohol :  the  toxin  mixed  with  numerous  impurities  is  carried  down  in  the 
precipitate.  Toxin  may  also  be  precipitated  by  ammonium  sulphate. 

Toxin  obtained  by  filtration  can  be  dried  in  vacuo  to  the  consistency  of  a 
dry  extract :  this  extract  is  soluble  in  water  and  contains  the  true  toxin 
mixed  with  a  very  large  proportion  of  impurities.  On  dialysis  the  watery 
solution  quickly  loses  the  mineral  salts  in  solution,  but  the  toxin  is  only 
removed  with  great  difficulty.  This  method  may  be  used  for  purifying  diph- 
theria toxin. 

The  toxic  content  of  diphtheria  toxin  is  considerable. 

1  c.c.  of  filtered  cultures  yields  O'Ol  gram  of  dry  residue :  thus,  if  0'005  c.c.  of 
filtered  culture  suffice  to  kill  a  guinea-pig  the  lethal  dose  of  the  dry  residue  is  f^ 
gram  (0'00005  gram)  and  of  this  small  quantity  the  greater  part  consists  of  mineral 
salts,  peptone  etc.  This  will  give  some  idea  of  how  infinitely  small  is  the  fatal 
dose  of  the  real  toxic  substance. 

Many  chemical  substances  alter  the  toxic  nature  of  diphtheria  toxin. 
For  instance,  toxin  is  destroyed  by  peptic  ferments,  while  alcohol,  acids,  and 
antipyrin  diminish  the  toxin  content.  Oxidizing  agents,  again,  are  remark- 
able for  the  capacity  they  exhibit  of  changing  its  character  :  thus,  hydrogen 
peroxide,  alkaline  hypochlorites  and  especially  iodine  and  iodine  terchloride 
lower  its  toxicity  considerably.  The  action  of  these  substances  is  turned  to 
practical  account  in  attenuating  toxin  which  is  to  be  used  for  the  immuniza- 
tion of  animals. 

4.  Vaccination. 

(i)  In  laboratory  animals  it  is  difficult  to  produce  immunity  by  repeated 
inoculation  of  very  small  doses  of  diphtheria  toxin  because  the  toxin  accumu- 
lates and  the  animals  become  cachectic  and  die. 

(a)  G.  Hoffmann  was  the  first  to  successfully  immunize  guinea-pigs.     He 
inoculated  them  first  with  cultures  attenuated  by  keeping,  and  later  with 
fully  virulent  cultures.     Subsequently,  Behring  and  Wernicke  employed  a 
similar  method. 

(b)  Frsenkel  immunized  guinea-pigs  by  inoculating  them  sub-cutaneously 
with  cultures  heated  for  an  hour  to  65°-70°  C.  :    altogether  the  animals 


rpp( 


IMMUNIZATION   OF  ANIMALS  263 


received  from  10-20  c.c.  of  these  cultures  at  various  times.     Immunity  was 
acquired  at  the  end  of  14  days. 

(c)  Behring  in  immunizing  guinea-pigs  and  rabbits  used  the  pleural  fluid 
obtained  from  guinea-pigs  which  had  succumbed  to  the  inoculation  of  virulent 
cultures.     Immunity  was  acquired  in  about  a  fortnight  after  the  inoculation 
of  the  vaccine,  but  the  results  were  very  inconstant. 

(d)  Behring  immunized  guinea-pigs  and  sheep  by  inoculating  them  with 
cultures  3  weeks  old  and  to  which  1  part  of  iodine  terchloride  had  been  added 
to  500  parts  of  culture.     A  few  c.c.  of  this  mixture  were  inoculated  into  an 
animal  and  then  10  days  or  so  later  a  second  inoculation  was  given  of  a 
culture  to  which  a  smaller  quantity  of  iodine  terchloride  had  been  added. 
Immunity  was  acquired  in  about  a  fortnight.     This  method  fails  in  the  case 
of  the  rabbit. 

(e)  Brieger,   Wassermann  and   Kitasato   conferred  immunity  on  guinea- 
pigs  by  inoculating  them  on  several  occasions  with  2  c.c.  of  a  culture  on 
thymus  broth  warmed  to  70°  C.  for  15  minutes.     But  this  method  is  not  so 
effective  as  the  iodine  terchloride  method  of  Behring. 

(ii)  Koux,  Nocard  and  Martin  succeeded  in  immunizing  various  animals 
(rabbits,  sheep,  goats,  cows,  horses)  by  inoculating  them  first  with  a  virulent 
toxin  mixed  with  Gram's  iodine  solution,  then  with  gradually  increasing 
doses  of  pure  toxin. 

A  rabbit,  for  example,  was  inoculated  sub-cutaneously  in  the  first  instance  with 
0*5  c.c.  of  the  following  mixture  which  was  prepared  immediately  before  use. 

Toxin  (Roux  and  Martin's  method), 2  vols. 

Gram's  iodine  solution,  .....         1  vol. 

The  injection  was  repeated  every  few  days  for  some  weeks,  then  the  proportion 
of  iodine  was  gradually  diminished  and  last  of  all  pure  toxin  was  inoculated.  The 
animals  were  weighed  at  frequent  intervals  and  if  they  showed  any  loss  of  weight 
the  inoculations  were  stopped  for  the  time  being,  otherwise  they  died  of  cachexia. 

Goats  and  cows  may  be  immunized  in  a  similar  manner,  but  these  animals  being 
very  susceptible  to  diphtheria  toxin,  very  small  doses  of  iodized  toxin  must  be 
used  for  the  initial  inoculations,  and  pure  toxin  should  only  be  given  when  the 
blood  shows  some  content  of  antitoxin.  It  should  be  borne  in  mind  that  pregnant 
animals  are  more  susceptible  to  diphtheria  toxin  than  non-pregnant  animals. 

Horses  stand  toxin  well  and  especial  interest  attaches  to  the  immunization 
of  these  animals  because  they  are  the  source  whence  antitoxin  for  thera- 
peutic purposes  is  derived. 

The  horses  selected  should  be  young  (6-9  years  old)  well  fed  and  free  from 
disease.  After  having  been  tested  with  mallein  to  exclude  a  possible  infection 
with  glanders  (vide  Glanders)  [and  with  tuberculin  to  exclude  tuberculosis] 
the  horse  is  inoculated  in  the  first  instance  with  a  small  quantity  of  a  virulent 
toxin  to  which  Grani's  iodine  has  been  added  :  at  subsequent  inoculations 
the  doses  are  gradually  increased,  and  after  the  eighth  inoculation  pure  toxin 
is  used  :  different  animals  vary  enormously  in  susceptibility,  and  care  should 
always  be  taken  that  the  dose  used  in  the  initial  experiment  shall  be  so  small 
that  no  violent  reaction  results,  as  this  might  imperil  the  steady  progress 
of  the  immunizing  process.  The  injections  should  be  made  sub-cutaneously 
into  the  neck  or  behind  the  shoulders. 

The  following  table  exhibits  an  actual  record  of  the  immunization  of  an  horse  by 
Roux  and  Nocard. 

Horse,  7  years  old  and  weighing  about  400  kg. 

The  iodized  toxin  contained  one-tenth  its  volume  of  Gram's  solution.  The  toxin  used 
killed  guinea-pigs  weighing  500  grams  in  48  hours  in  doses  of  O'l  c.c.  The  injections 
were  made  beneath  the  skin  of  the  neck  or  behind  the  shoulder. 

1st  day  of  experiment.     Injection  of  0*25  c.c.  of  an  iodized  toxin.     No  local 
nor  general  reaction. 


264  THE  DIPHTHERIA   BACILLUS 

2nd  day  of  experiment.  Injection  of  0*5  c.c.  of  an  iodized  toxin.  No  local 
nor  general  reaction. 

4th,  6th,  and  8th  days  of  experiment.  Injection  of  0*5  c.c.  of  an  iodized  toxin. 
No  local  nor  general  reaction. 

13th  and  14th  days  of  experiment.  Injection  of  1  c.c.  of  an  iodized  toxin.  No 
reaction. 

17th  day  of  experiment.  Injection  of  0'25  c.c.  of  a  pure  toxin.  Slight  oedema. 
No  rise  of  temperature. 

22nd  day  of  experiment.  Injection  of  1  c.c.  of  a  pure  toxin.  Slight  oedema. 
No  rise  of  temperature. 

23rd  day  of  experiment.  Injection  of  2  c.c.  of  a  pure  toxin.  Slight  oedema. 
No  rise  of  temperature. 

25th  day  of  experiment.  Injection  of  3  c.c.  of  a  pure  toxin.  Slight  oedema. 
No  rise  of  temperature. 

28th  day  of  experiment.  Injection  of  5  c.c.  of  a  pure  toxin.  Slight  oedema. 
No  rise  of  temperature. 

30th,  32nd,  and  36th  days  of  experiment.  Injection  of  5  c.c.  of  a  pure  toxin. 
Slight  oedema.  No  rise  of  temperature. 

39th  and  41st  days  of  experiment.  Injection  of  10  c.c.  of  a  pure  toxin.  Slight 
oedema.  No  rise  of  temperature. 

43rd,  46th,  48th  and  50th  days  of  experiment.  Injection  of  30  c.c.  of  a  pure 
toxin.  Fairly  well  marked  oedema  which  vanished  in  24  hours. 

53rd  day  of  experiment.  Injection  of  60  c.c.  of  a  pure  toxin.  Fairly  well 
marked  oedema  which  vanished  in  24  hours. 

57th,  63rd.  65th,  and  67th  days  of  experiment.  Injection  of  60  c.c.  of  a  pure 
toxin.  Fairly  well  marked  oedema  which  vanished  in  24  hours. 

72nd  day  of  experiment.  Injection  of  90  c.c.  of  a  pure  toxin.  Fairly  well 
marked  oedema  which  vanished  in  24  hours. 

80th  day  of  experiment.  Injection  of  250  c.c.  of  a  pure  toxin.  Fairly  well 
marked  oedema  which  vanished  in  24  hours. 

This  horse  therefore  had  received  in  2  months  and  20  days  800  c.c.  of  toxin,  with- 
out showing  any  symptoms  other  than  a  transient  local  oedema,  some  loss  of  appetite 
and  a  rise  of  temperature  of  about  1°  C.  on  the  evenings  following  the  larger  injec- 
tions. The  animal  was  bled  on  the  87th  day  and  was  inoculated  into  the  jugular 
vein  with  200  c.c.  of  toxin  without  showing  any  reaction. 

Vaccinated  horses  withstand  equally  well  enormous  doses  (many  hundred  cubic 
centimetres)  of  living  cultures. 

As  has  been  pointed  out  some  horses  are  more  susceptible  to  diphtheria  toxin 
than  others,  and  in  the  more  susceptible  individuals  an  extensive,  firm  oedema 
lasting  many  days  may  follow  inoculation,  and  in  some  cases,  the  horse  may  sweat 
and  show  a  marked  rise  of  temperature. 

Occasionally  a  highly  immunized  horse  will  die  of  paralysis  1-3  weeks  after  the 
last  inoculation  of  toxin. 

With  the  very  powerful  toxins  at  present  in  use  immunization  should  be 
carried  out  still  more  carefully.  With  a  toxin  containing  200  units  of  toxin 
per  c.c.  horses  should  be  inoculated  three  times  a  week  for  6  weeks,  with  a 
mixture  of  toxin  and  Gram's  solution  (commencing  with  a  mixture  consisting 
of  2  parts  Gram's  solution  and  1  part  toxin),  then  with  toxin  alone  in  pro- 
gressively increasing  doses  :  the  initial  dose  being  0'5  c.c.  and  the  final 
inoculation  200  c.c. 

When  small  doses  are  inoculated  at  frequent  intervals  the  antitoxic  content 
of  the  serum  is  greater  than  when  large  doses  are  given  at  longer  intervals 
(Roux). 

To  maintain  horses  in  a  state  of  immunization  it  is  necessary  to  inoculate 
a  dose  of  toxin  from  time  to  time  :  this  may  be  done  in  different  ways. 

1.  After  bleeding  a  horse  300-500  c.c.  of  culture  may  be  inoculated  at 
intervals  of  20  or  25  days  into  the  jugular  vein. 

2.  At  the  Pasteur  Institute  Martin  inoculates  beneath  the  skin  of  the 
shoulder,  13  days  after  bleeding  the  animal,  300  c.c.  of  toxin  ;    and  4  days 
later  on  the  opposite  side — also  sub-cutaneously — a  further  500  c.c.     The 
horse  may  be  bled  again  a  week  after  the  last  inoculation. 


DIPHTHERIA  ANTITOXIN 


265 


3.  Another  method  is  to  inoculate  sub-cutaneously  every  2  or  3  days  for 
3  weeks  quantities  of  toxin  increasing  from  25-150  c.c.  until  about  a  litre 
has  been  injected.  The  animal  is  bled  12  days  after  the  last  inoculation 
of  toxin. 

(iii)  Horses  may  also  be  immunized  by  inoculating  them  with  a  mixture 
of  antidiphtheria  serum  and  toxin  (Babes,  Madsen  and  Dreyer,  Park).  The 
yield  of  antitoxin  is  good  and  the  method  is  more  rapid  than  the  iodine 
terchloride  method  (Park). 

The  following  table  shows  the  details  of  an  experiment  by  Martin. 
Inoculations  twice  a  week.     Toxin  killed  guinea-pigs  weighing  500  grams  in  36  hours 
in  doses  of  O'l  c.c.     Antidiphtheria  serum  contained  200  units  per  c.c. 

1st  inoculation,  -  25  c.c.  serum  +  25  c.c.  toxin. 


2nd 

3rd 

4th 

5th 

6th 

7th 

8th 

9th 

10th 

llth 

12th 

13th 

14th 

15th 


10     „          „     +   25 

25  c.c.  pure  toxin. 

40 

60 

80 
100 
150 
200 
250 
300 
350 
400 
450 
500 


5.  Serum  therapeutics. 

Antitoxin. 

Behring  and  Kitasato  in  1890  were  the  first  to  demonstrate  the  antitoxic 
properties  of  the  blood  of  animals  immunized  against  diphtheria. 

These  observers  found  that  the  blood  of  immunized  animals  had  the  property  of 
destroying  diphtheria  toxin  both  in  vivo  and  in  vitro  ;  that  this  property  was  also 
present  in  the  serum  of  blood  deprived  of  all  cellular  elements  ;  and  that  the  serum 
was  both  therapeutic  and  prophylactic  when  used  on  rabbits  and  guinea-pigs  intoxi- 
cated with  diphtheria  toxin  or  inoculated  with  living  diphtheria  bacilli. 

Having  established  these  facts  Behring,  Ehrlich,  and  their  collaborators  turned 
their  attention  to  the  application  of  antidiphtheria  serum  to  the  treatment  of 
human  diphtheria  (Behring,  Ehrlich,  Boer,  Wassermann,  Rossel).  But.  the  serum 
treatment  of  diphtheria  did  not  become  an  accomplished  fact  in  medical  practice 
until  after  the  Congress  of  Hygiene  at  Buda-Pesth  in  1894  when  Roux  and  Martin 
communicated  a  summary  of  the  work  they  had  carried  out  during  the  years  1891-4. 

(a)  Preparation  of  the  serum. 

The  horse  is  chosen  as  the  source  of  antitoxin  for  these  reasons,  viz.  : — 
Horse  serum,  even  in  large  doses,  is  innocuous  to  man  and  to  the  lower 
animals  ;  horses  withstand  the  action  of  diphtheria  toxin  very  much  better 
than  other  animals  ;  lastly,  very  large  quantities  of  serum  are  available 
(Roux  ;  Nocard  and  Martin). 

The  immunization  of  the  horse  which  is  carried  out  as  described  above 
generally  occupies  about  3  months.  In  practice  toxin  is  inoculated  in  gradu- 
ally increasing  doses  until  some  1000-1500  c.c.  have  been  administered  :  the 
final  inoculations  should  consist  of  quantities  of  150-200  c.c. 

The  animal  is  bled  8-10  days  after  the  date  of  the  last  inoculation  :  about 
6  litres  of  blood  are  withdrawn  and  a  further  quantity  may  be  taken  a  few 
days  later.  It  is  best  to  bleed  the  horse  from  the  jugular  vein  according  to 


266  THE  DIPHTHERIA  BACILLUS 

the  directions  given  on  pp.  49  and  50  (Nocard's  method  and  Latapie's 
apparatus).  Six  litres  of  blood  yield  nearly  4  litres  of  serum. 

The  horse  is  maintained  in  a  state  of  immunization  by  the  inoculation  of 
toxin  from  time  to  time. 

When  several  animals  have  been  immunized  it  is  highly  desirable  that  the  serum 
of  the  various  horses  should  be  mixed  ;  by  doing  this  a  product  is  obtained  of  which 
the  antitoxin  content  is  uniform.  Moreover,  the  serum  of  some  horses  is  liable  to 
provoke  erythematous  rashes  when  used  in  the  human  subject,  which  though  harm- 
less are  nevertheless  irritating,  and  by  mixing  different  serums  this  inconvenience 
may  be  minimized. 

For  the  purpose  of  storing  serum  it  is  distributed  with  aseptic  precautions 
in  small  sterile  bottles  stoppered  with  sterile  india-rubber  plugs  and  kept 
in  the  dark. 

Serum  prepared  under  strictly  aseptic  precautions  may  be  kept  in  these 
climates  many  months  in  a  sterile  condition  without  losing  any  of  its  anti- 
toxic properties.  Occasionally  the  serum  after  bottling  becomes  distinctly 
cloudy,  but  this  is  of  no  importance  with  respect  either  to  the  purity  or 
efficacy  of  the  serum.  A  deposit  is  less  likely  to  occur  and  the  keeping  pro- 
perty of  the  serum  is  better  assured  if,  immediately  after  filling,  the  bottles 
are  heated  for  an  hour  at  57°  C.  in  a  water  bath.  This  degree  of  heat  has  no 
effect  upon  the  properties  of  the  serum. 

Dried  serum. — Serum  may  be  dried  by  evaporation  in  vacuo.  Just  before 
use  the  dried  serum  is  dissolved  in  eight  or  ten  times  its  volume  of  sterile  water ; 
this  solution  frequently  gives  rise  to  a  local  but  transient  swelling  which  is  not  the 
case  with  liquid  serum.  In  these  latitudes  liquid  serum  should  always  be  admini- 
stered in  preference  to  the  dry  product :  the  value  of  the  latter  is  apparent  in  warm 
climates  where  liquid  serum  quickly  loses  its  properties. 

Antitoxic  milk. — The  milk  of  immunized  females  possesses  antitoxic  properties 
(Ehrlich).  This  fact  however  is  merely  of  theoretical  interest  because  the  extreme 
dilution  of  the  antitoxin  in  the  milk  renders  the  latter  incapable  of  being  used  in 
practice. 

Still,  milk  containing  antitoxin  may  be  condensed  to  a  sufficiently  small  volume 
to  allow  of  laboratory  experiments  being  conducted  with  it  (Wassermann).  The 
milk  of  cows  or  goats  can  be  used  ;  for  experimental  purposes  it  is  scarcely  possible 
to  get  a  milk  with  a  preventive  strength  of  one-fifth  (vide  infra,  p.  268). 

(b)  Properties  of  the  serum. 

The  serum  of  immunized  animals  is  antitoxic,  that  is  to  say  if  the  serum  be 
mixed  with  toxin  in  suitable  quantities  the  mixture  is  harmless  on  inoculation 
into  animals. 

This  property  of  the  serum  is  due  to  a  special  substance  known  as  Antitoxin,  the 
nature  of  which  is  as  little  understood  as  is  the  nature  of  toxin  :  like  toxin,  antitoxin 
is  altered  by  heat,  precipitated  by  alcohol  and  carried  down  with  the  precipitates 
formed  in  liquids  which  contain  it  in  solution.  In  the  living  body  it  is  formed  in 
response  to  the  absorption  of  toxin ;  "  under  the  influence  of  toxin,  certain  cells 
of  the  living  body  acquire  a  new  and  persistent  secretory  property  "  (Salomonsen 
and  Madsen). 

Antitoxin  saturates  toxin  both  in  vivo  and  in  vitro  (p.  224)  :  it  has  both 
prophylactic  and  curative  properties  :  a  guinea-pig  inoculated  with  an  adequate 
dose  of  serum  can  withstand  the  subsequent  inoculation  of  such  a  quantity 
of  toxin  as  would  be  sufficient  to  kill  with  certainty  a  non-inoculated  guinea- 
pig.  Even  if  the  toxin  be  inoculated  first  and  the  serum  not  until  several 
hours  later,  the  animal  will  be  protected.  Immunity  is  rapidly  produced 
but  is  short-lived  :  in  a  few  days  or  weeks  it  has  entirely  disappeared.  The 
amount  of  serum  necessary  to  cure  an  animal  inoculated  with  toxin  depends 
upon  many  factors  :  among  others  upon  the  weight  of  the  animal,  the  amount 


STANDARDIZATION   OF  ANTITOXIN  267 

and  toxicity  of  the  toxin  used  and  upon  the  antitoxic  strength  of  the  serum. 
It  is  very  important  to  know  the  antitoxic  strength  of  the  serum  used,  and 
rules  have  been  devised  by  which  this  may  be  determined  (vide  infra). 

Antitoxic  serum  is  not  bactericidal  and  contains  no  immune  body  (sensibili- 
satrice)  ;  it  has  some  power  of  agglutinating  the  bacillus  but  only  in  a  feeble 
and  inconstant  manner.  For  instance,  it  may  agglutinate  in  dilutions  of 
1  in  10  and  1  in  20  and  the  agglutination  is  sometimes  visible  to  the  naked 
eye,  flocculi  falling  to  the  bottom  of  the  tube  (Nicolas)  ;  on  the  other  hand 
agglutination  is  often  absent.  The  serum  also  of  patients  suffering  from  diph- 
theria sometimes  shows  some  slight  property  of  agglutination  (Bruno). 

Bacilli  recently  isolated  from  the  living  subject  are  often  unaffected  or  but  slightly 
affected  by  the  specific  agglutinin  ;  prolonged  sojourn  in  artificial  culture,  however, 
seems  to  develop  or  increase  the  power  of  being  agglutinated.  It  is  important  in 
testing  the  agglutinating  properties  to  use  a  good  emulsion  of  bacilli,  and  for  this 
purpose  a  culture  in  2  per  cent,  glucose  broth,  which  is  generally  cloudy,  is  suitable. 
Martin  recommends  collecting  the  bacilli  from  flasks  which  have  been  used  for  toxin 
preparation  and  heating  them  to  100°  C.  For  use,  shake  up  these  bacilli  well  with 
normal  saline  solution  and  let  the  emulsion  stand :  a  large  number  of  the  bacilli 
will  deposit,  but  the  supernatant  liquid  remains  cloudy  permanently  and  serves 
very  well  for  the  agglutination  reaction. 

Antitoxic  serum  is  both  prophylactic  and  therapeutic  in  the  case  of  animals 
inoculated  with  a  living  culture  of  the  diphtheria  bacillus  :  the  therapeutic 
properties  are  exhibited  even  if  the  serum  be  not  administered  until  12  or  18 
hours  after  the  virus. 

In  the  case  of  a  guinea-pig  which  has  been  inoculated  with  a  living  culture  on 
the  mucous  membrane  of  the  trachea  or  vulva,  the  inoculation  of  serum,  even  though 
it  be  administered  before  the  virus,  will  not  prevent  the  formation  of  the  charac- 
teristic false  membrane,  but  does  entirely  prevent  symptoms  of  intoxication  or  of 
disturbance  of  the  general  health  of  the  animal :  and  further  the  false  membrane 
becomes  detached  on  the  second  day  and  the  infected  surface  commences  to  heal. 
If  instead  of  being  inoculated  before  the  living  virus,  the  serum  be  administered 
after  the  false  membrane  has  formed,  it  leads  to  the  disappearance  of  the  oedema 
and  swelling  in  a  few  hours  and  after  two  days  to  the  casting  off  of  the  false  membrane. 

The  false  membranes  formed  in  the  trachea  of  the  rabbit  as  the  result  of  infecting 
the  mucous  membrane  with  a  mixture  of  streptococci  and  B.  diphtheric?  are  not  so 
readily  affected  by  antitoxic  serum.  In  such  a  case  5,  and  even  10  c.c.  of  antitoxin 
are  insufficient  to  save  the  life  of  the  animal :  but  Roux  and  Martin  have  been  able 
to  effect  a  cure  in  parallel  cases  by  repeating  the  inoculation  of  antitoxin  several 
times. 

Roux  and  Martin  have  tested  the  value  in  these  cases  of  mixing  antistreptococcal 
and  antidiphtheria  serums,  but  with  only  moderately  successful  results  (vide  The 
Streptococci). 

(c)  The  standardization  of  antitoxin. 

1.  Behring  estimated  the  antitoxic  content  of  an  antiserum  in  terms  of 
the  amount  of  the  serum  necessary  to  immunize  1  gram  weight  of  animal 
against  the  minimal  fatal  dose  of  toxin  inoculated  12  hours  after  the  serum. 

Thus,  for  example,  if  1  c.c.  of  a  serum  immunized  1  kg.  weight  of  guinea-pig 
against  the  inoculation  12  hours  later  of  the  smallest  dose  of  toxin  which  would  kill 
a  control  animal  of  the  same  weight  within  a  given  time  the  serum  was  said  to  have 
a  strength  of  f^Q.  This  method  of  titration  is  not  very  exact  but  it  has  the 
advantage  of  being  simple. 

2.  Behring  then  altered  his  test  inoculation.     Instead  of  toxin  he  used 
living  bacilli  and  measured  the  value  of  the  serum  against  an  infection  and 
not  against  an  intoxication.    The  unit  of  serum,  now,  was  the  amount  necessary 
to  immunize  5,000  grams  of  guinea-pig  (or  10  guinea-pigs  of  500  grams  each) 
against  the  inoculation  24  hours  later  of  ten  times  the  amount  of  a  forty -eight- 


268  THE  DIPHTHERIA  BACILLUS 

hour  old  culture  of  the  diphtheria  bacillus  which  was  certainly  fatal  to  control 
animals. 

[Thus  if  0*01  c.c.  of  a  serum  would  immunize  a  guinea-pig  weighing  500  grams 
against  10  times  the  lethal  dose  of  a  48  hour  old  culture  of  a  virulent  diphtheria 
bacillus,  it  follows  that  O'l  c.c.  would  be  required  to  immunize  5,000  grams.  That 
amount  then  was  the  unit  and  1  c.c.  of  the  serum  would  contain  10  units.] 

3.  Ehrlich  adopted  another  unit  of  measure.     The  unit  of  antitoxin  (I.E.) 
is  the  amount  of  antitoxin  necessary  to  neutralize  100  minimal  lethal  doses 
of  normal  toxin  (i.e.  of  a  toxin  which  is  fatal  to  guinea-pigs  in  doses  of  0*1  c.c.). 
Thus  if  O'Ol  c.c.  of  a  given  serum  neutralizes  100  fatal  doses  of  toxin  that 
serum  is  said  to  contain  100  antitoxic  units  per  c.c. 

It  was  difficult  to  get  comparable  results  by  this  method  because  the 
toxicity  of  a  given  toxin  diminishes  with  the  lapse  of  time.  On  the  other 
hand  the  antitoxin  content  of  a  carefully  standardized  serum  dried  in  vacua 
and  without  heating  is  known  to  remain  constant  almost  indefinitely. 
Ehrlich  therefore  having  determined  an  antitoxic  unit  (as  above)  prepares  a 
glycerin  solution  of  the  serum  containing  17  units  per  c.c.  This  preparation 
is  now  used  in  antitoxin  laboratories  as  the  standard  for  testing  serums. 
One  c.c.  of  Ehrlich's  glycerin  solution  diluted  with  16  c.c.  of  water  gives  a 
solution  containing  1  unit  of  antitoxin  per  c.c. 

It  is  then  easy  to  titrate  a  toxin  against  the  standard  antitoxin  and  after 
standardizing  the  toxin  very  carefully  the  latter  is  used  to  titrate  the  anti- 
toxin under  investigation. 

4.  The    French    method    of   standardizing    antitoxin. — Roux    prefers     to 
standardize  antitoxin  according  to  its  preventive  strength  (pouvoir  preventif). 
The  preventive  strength  is  the  numerical  ratio  between  the  weight  of  a  given 
animal — guinea-pig — in  grams  and  the  amount  of  serum  necessary  to  save 
its  life  if  it  be  inoculated  12  hours  later  with  0*5  c.c.  of  a  young  and  highly 
virulent  culture.     For  example,  if  the  guinea-pig  weigh  500  grams  and  O'l  c.c. 
of  serum  has  to  be  inoculated  to  protect  it  against  the  subsequent  inoculation 
of  culture  the  preventive  strength  is  •££§,  and  the  serum  is  said  to  be  active 

in        1       1 

111    5,000' 

In  practice  the  Ehrlich  and  Roux  methods  may  usefully  be  controlled 
against  each  other  ;  for  instance,  a  serum  is  said  to  contain  100  antitoxic 
units  per  c.c.  and  to  have  a  preventive  strength  of  50.Q0(j.  It  must  be  borne 
in  mind  however,  that  the  maximum  of  preventive  strength  may  not  coincide 
with  the  maximum  of  antitoxic  strength  (Martin,  Momont  and  Prevot). 

A  serum  which  has  a  preventive  strength  of  50  *00  is  suitable  for  the 
treatment  of  diphtheria  in  man,  but  a  more  powerful  serum  (70  *  00  or  even 
100^000)  is  easily  prepared.  The  serum  made  at  the  Pasteur  Institute  with 
the  older  toxins  contained  100  antitoxic  units  per  c.c.  and  had  a  preventive 
strength  of  50  QOO  ;  but  at  the  present  time,  using  toxins  prepared  by  growing 
the  bacillus  in  Martin's  broth  which  are  ten  times  stronger  than  the  older 
toxins,  the  serum  supplied  contains  at  least  200-300  antitoxic  units  per  c.c. 
and  has  a  preventive  strength  of  7-00700  0- 

(d)  Serum  therapeutics. 

The  application  of  serum  therapy  to  the  treatment  of  diphtheria  in  the 
human  subject  has  yielded  results  which  might  have  been  expected  from 
animal  experiment.  The  serum  therapy  of  diphtheria  is  one  of  the  most 
striking  successes  of  modern  therapeutics. 

1  Animals  treated  in  this  way  do  not  survive  indefinitely  but  die  generally  in  from 
1-6  months  (Roux). 


SERUM  THERAPEUTICS  269 


The  serum  is  inoculated  in  doses  of  10-40  c.c.,  either  in  one  dose  or  at 
different  times  according  to  the  severity  of  the  disease  :  the  technique  of 
administration  need  not  be  dealt  with  here. 

The  serum  has  been  used  as  a  prophylactic  during  epidemics  of  diphtheria  ; 
the  immunity  so  produced  is  only  of  short  duration  and  varies  from  3-6  weeks. 
The  dose  to  be  used  as  a  prophylactic  should  be  5-10  c.c.  (Roux). 

6.  Agglutination. 

Antitoxic  serum  obtained  by  the  inoculation  of  toxin  possesses,  as  has  been 
pointed  out,  no  agglutinating  properties  and  contains  no  immune  body 
(substance  sensibilisa trice).  By  the  inoculation  of  the  bacilli  themselves  a 
serum  containing  both  agglutinins  and  immune  body  can  be  obtained .  Wasser- 
mann,  Bandi,  Martin,  thought  that  such  a  serum  might  be  of  use  in  clinical 
practice  to  facilitate  the  disappearance  of  diphtheria  bacilli  from  the  pharynx 
of  those  cases  in  which  it  remained  an  unduly  long  time  even  after  the  use  of 
antitoxin. 

(i)  Wassermann  inoculated  into  the  veins  of  a  rabbit  an  extract  of  bacilli 
to  which  antitoxin  had  been  added  to  neutralize  the  toxin.  After  several 
inoculations  the  serum  of  the  rabbit  precipitated  the  bacillary  extract  and 
agglutinated  the  diphtheria  bacillus. 

It  would  not  appear  to  be  true,  as  Wassermann  thought,  that  this  serum  can  be 
used  to  differentiate  the  pseudo- diphtheria  from  the  diphtheria  bacillus.  As  Lip- 
stein  points  out,  a  serum  obtained  by  the  inoculation  of  a  given  strain  of  bacilli  may 
agglutinate  that  strain  in  high  dilution,  while  having  no  effect  whatever  on  other 
strains. 

(ii)  Lipstein  obtained  a  serum  which  agglutinated  the  diphtheria  bacillus 
in  a  dilution  of  1  in  1,000  by  inoculating  into  the  peritoneal  cavity  of  a  rabbit 
first  a  mixture  of  dead  bacilli  and  antitoxin  and  later  a  mixture  of  living 
bacilli  and  antitoxin.  This  serum  has  no  prophylactic  properties. 

(iii)  Bandi  inoculated  a  dog  sub-cutaneously  several  times  during  the 
course  of  a  month  with  sensitized  diphtheria  bacilli — bacilli,  that  is,  which  had 
been  treated  with  antitoxin  and  had  then  been  washed  and  centrifuged  to 
remove  any  excess  of  serum.  This  observer  obtained  a  serum  which  besides 
possessing  agglutinating  and  sensitizing  properties  was  also  feebly  antitoxic 
(15  units  per  c.c.).  He  had  good  results  in  seven  cases  of  diphtheria  which 
he  treated  with  the  serum. 

(iv)  Martin  also  prepared  a  serum  which  exhibited  agglutinating,  sensitizing 
and  immunizing  properties.  The  serum  was  obtained  from  a  horse  by 
inoculating  it  sub-cutaneously,  intra-peritoneally,  or  better  into  the  veins 
with  bacilli  which  had  been  heated  to  100°  C.  for  an  hour.  Martin  treated 
a  number  of  cases  of  diphtheria  locally  with  this  serum.  Repeated  applica- 
tion to  the  false  membranes  gave  very  little  result — some  diminution  of  pain 
— but  by  making  it  into  pastilles  with  gum  and  so  ensuring  a  more  prolonged 
contact  with  the  membrane  the  results  were  more  satisfactory.  Under  these 
conditions  the  false  membranes  swelled  up,  became  softened  and  were  soon 
detached.  Cultures  showed  a  rapid  and  marked  diminution  in  the  number 
of  bacilli. 


SECTION  IV.— DETECTION,   ISOLATION,   AND   IDENTIFICATION  OF 
THE   DIPHTHERIA   BACILLUS. 

The  diagnosis  of  diphtheria  is  often  impossible  by  clinical  methods  alone  ; 
hence  in  practice  the  nature  of  the  infecting  agent  in  all  cases  of  croup  and 


270  THE  DIPHTHERIA  BACILLUS 

sore  throat,  especially  if  any  trace  of  a  false  membrane  be  present,  has  to  be 
determined  by  bacteriological  investigation. 

By  this  means  it  can  be  ascertained  whether  any  given  case  of  croup  or 
sore  throat  be  the  result  of  an  infection  with  the  diphtheria  bacillus  either 
alone  or  in  association  with  other  organisms.  If  a  diphtheria  bacillus  be 
found,  its  virulence  should  be  ascertained  ;  but  this  is  not  a  matter  of  great 
importance  in  practice,  because  as  a  rule  it  may  be  assumed  that  the  long 
bacillus  is  the  most  virulent  of  all  the  varieties  of  the  diphtheria  bacillus — 
and  so  the  virulence  of  the  strain  under  examination  may  be  gauged  from  the 
relative  number,  or  entire  absence,  of  such  forms — and  because  from  the 
point  of  view  of  treatment  the  mere  fact  that  the  diphtheria  bacillus  has 
been  found  demands  the  administration  of  antitoxin. 

When  dealing  with  a  case  of  sore  throat  three  investigations  are  necessary 
before  the  bacteriological  examination  can  be  said  to  be  scientifically  complete. 
First  films  from  the  inflamed  surface  must  be  examined,  then  cultures  must 
be  sown  with  the  material  from  the  throat,  and  lastly  the  causal  organism 
must  be  isolated  and  injected  into  an  animal.  In  clinical  work,  however, 
the  two  former  investigations  are  all  that  is  required  and  these  occupy  24 
hours  at  the  outside. 

1.  Collection  of  the  material. 

Remove  the  false  membrane  on  a  small  piece  of  absorbent  cotton-wool 
fixed  in  a  pair  of  pressure  forceps  :  in  those  cases  in  which  the  membrane  is 
very  adherent  it  is  better  to  tear  it  off  with  the  forceps.  For  the  collection 
of  material  in  the  ordinary  way  it  is  convenient  to  have  a  small  cotton-wool 
plug  fixed  on  the  end  of  a  metal  rod  ;  this  is  placed  in  a  test-tube  which  is  then 
plugged  with  wool  and  the  whole  sterilized  in  the  hot  air  sterilizer.  If  there 
be  no  membrane  scrape  the  surface  of  the  tonsils  or  pharynx  with  a  small 
platinum  or  nickel  spatula. 

When  it  is  desired  to  send  a  fragment  of  false  membrane  to  a  laboratory  situated  at  a 
distance,  place  it  in  a  small  sterilized  tube  plugged  with  wool ;  or  wrap  it  in  a  piece  of 
thin  cloth  which  has  been  passed  through  boiling  water  and  then  place  it  in  a  new  glass 
tube  carefully  plugged. 

Most  laboratories  send  the  necessary  apparatus  for  the  collection  of  material  to  prac- 
titioners. A  convenient  form  is  that  which  contains  two  sterilized  plugs  in  test-tubes, 
a  sterile  tube  for  false  membrane  and  a  small  spatula,  as  well  as  two  tubes  of  serum. 

2.  Methods  of  examination. 

A.  Microscopical  examination  of  the  fresh  material.— This  part  of  the 
investigation  is  of  considerable  importance. 

Before  using  the  false  membrane  for  bacteriological  examination  press  it 
lightly  between  sterile  filter-paper  to  blot  up  any  mucus  which  may  be  present 
on  the  surface. 

1.  Prepare  films  with  small  portions  of  the  exudate,  and  stain  with  Roux's 
blue,  wash  and  dry.  Examine  with  an  oil  immersion  lens.  [Cobbett's  method 
(p.  252)  is  recommended  as  giving  more  characteristic  appearances.] 

The  absence  of  the  diphtheria  bacillus  must  not  be  assumed  if  the  micro- 
scopical examination  be  negative  as  it  is  a  well  known  fact  that  it  often  passes 
unrecognized  when  mixed  with  a  number  of  other  organisms  (Martin). 

Should  bacilli  resembling  diphtheria  bacilli  be  found  in  the  preparation, 
the  diagnosis  may  be  advanced  a  stage  by  staining  other  films  by  Gram's 
method.  The  diphtheria  bacillus  is  gram-positive,  and  a  certain  number 
of  bacilli  frequently  found  in  the  mouth  and  morphologically  resembling  it, 
but  gram-negative,  can  by  this  means  be  excluded. 

Cultures  must  be  sown  in  every  case. 


BACTERIOLOGICAL  DIAGNOSIS  271 

One  or  two  important  inferences  may  be  drawn  from  the  result  of  micro- 
scopical examination,  when  positive.  As  a  rule,  the  finding  of  numerous 
bacilli  of  the  "  long  "  type  denotes  a  severe  infection,  and  a  similar  inference 
may  be  drawn  if  streptococci  are  found  associated  with  the  specific  micro- 
organism. On  the  other  hand  the  presence  of  the  Brisou  coccus  generally 
indicates  a  mild  infection. 

2.  Sections. — Harden  in  alcohol,  embed  in  paraffin  and  cut  sections  per- 
pendicularly to  the  surface  of  the  membrane.  For  staining,  Gram's  method 
with  double  counterstain  gives  very  good  preparations. 


•',  ,•?*„•,  «•' 

•>  »   \ 


*+  **»  .   I, 


FIG.  171. — Section  of  tracheal  membrane  from  a  case  of  diphtheria,  showing 
diphtheria  bacilli  (Eosin  and  methylene  blue),  oc.  2 ;  obj.  Tl5th,  Zeiss. 

From  an  histological  study  of  diphtheria  membranes  it  can  be  shown  that  they 
are  made  up  of  three  layers  :  the  deepest  layer — that  next  the  body  surface 
(mucous  membrane  or  skin) — consists  of  a  network  of  fibrin  enclosing  epithelial 
cells  and  leucocytes  :  the  middle  layer  is  made  up  of  granular  fibrin  with  but  few 
cellular  elements,  while  the  most  superficial  layer  consists  almost  entirely  of  micro- 
organisms ;  the  bacilli,  many  of  which  are  swollen  at  the  ends,  are  arranged  in 
masses  parallel  to  one  another,  and  side  by  side  with  the  diphtheria  bacillus  are 
found  the  organisms  associated  with  it. 

B.  Cultures. — Cultures  should  be  sown  on  coagulated  blood  serum.  If 
however  this  medium  be  not  available,  white  of  egg  may  be  used  instead. 

The  various  serum  media  of  Lcefner,  Tochtermann,  Joos,  etc.,  give  no  better 
results  than  coagulated  blood  serum  while  they  have  the  disadvantage  of  needlessly 
complicating  the  technique.  [Many  observers,  however,  state  that  Lceffler's  serum 
(p.  52)  is  by  far  the  best  medium  for  the  cultivation  of  the  diphtheria  bacillus. 

[Lorrain  Smith  prepared  a  transparent  serum  medium  by  adding  O'l  per  cent,  to 
0*15  per  cent,  of  caustic  soda  to  ox  serum  and  heating  the  mixture  at  120°  C. 

[Cobbett  added  about  1  c.c.  of  a  10  per  cent,  solution  of  caustic  soda  to  100  c.c.  of 
ox  or  horse  serum  and  after  thoroughly  mixing  added  1  per  cent,  of  glucose  and 
sterilized  at  a  temperature  of  87°  C.  ] 

[Coplans  has  recently  introduced  the  following  medium  for  the  routine  recognition 
of  the  diphtheria  bacillus  : 

Sheep's  serum, 75     parts. 

Broth,    -  25 

Glucose, 0'5  per  cent. 

KCNS., 1 

CaCl2, 1 

1  per  cent,  aqueous  solution  of  neutral  red,     -         -         -  0*25      „ 


272  THE  DIPHTHERIA  BACILLUS 

[The  medium  is  adjusted  so  that  on  coagulation  the  reaction  is  but  faintly  alkaline. 
When  throat  swabs  are  sown  on  surface  slopes  and  incubated  at  37°  C.  for  18  hours, 
colonies  of  the  diphtheria  bacillus  appear  almost  invariably  to  yield  a  bluish-pink 
tint,  with  diffusion  of  like  tint  through  the  medium  ;  with  Hofmann's  bacillus  the 
growth  is  yellowish  and  alkaline  with  diffusion  of  a  yellowish  tint.  Staphylococci 
usually  yield  a  straw-coloured  raised  growth  with  discrete  colonies,  but  certain 
varieties  produce  either  discrete  pink  colonies  with  strictly  local  diffusion  of  tint 
into  the  medium  ;  and  again,  other  varieties,  more  especially  such  as  are  derived 
from  the  throats  of  adults,  yield  acid  with  pink  colouration  of  the  medium  in  varying 
intensity.  The  colonies  of  torulse  are  usually  raised  and  straw-coloured  but  they 
may  be  brownish  or  red.] 

Sow  a  tube  of  blood  serum  with  a  small  piece  of  membrane  held  in  a  platinum 
loop  and  after  rubbing  it  all  over  the  surface  of  the  serum  and  without  recharg- 
ing the  loop  sow  two  other  serum  tubes  (p.  82  B 1).  In  the  absence  of  membrane 
the  spatula  or  cotton-wool  swab,  as  the  case  may  be,  which  has  previously 
been  applied  to  the  tonsils  or  pharynx  is  rubbed  over  the  surface  of  the 
serum.  Should  the  cotton-wool  swab  be  dry  on  arrival  at  the  laboratory 
wash  it  in  a  little  sterile  water  and  then  use  the  latter  for  sowing  cultures. 
Dried  membranes  should  similarly  be  softened  in  sterile  water  before  being 
sown. 

Incubate  the  cultures  at  37°  C.  and  examine  about  20  hours  later  :  colonies 
of  the  diphtheria  bacillus  are  easily  recognized  at  this  stage  ;  some  cocci 
indeed  produce  a  very  similar  growth  but  it  is  moister  and  more  homogeneous 
than  that  of  the  diphtheria  bacillus.  A  mere  naked  eye  examination  of  the 
growth  is  however  insufficient,  and  must  always  be  supplemented  by  micro- 
scopical examination.  If  examination  of  the  cultures  be  delayed  beyond 
24  hours  difficulty  may  arise  from  the  development  of  micro-organisms  which 
are  either  associated  with  the  diphtheria  bacillus  or  which  are  present  as  an 
impurity.  Select  the  culture  which  shows  the  greatest  number  of  discrete 
colonies. 

[In  examining  cultures  sown  with  swabs  from  infected  throats  Cobbett  picks  off 
single  colonies  one  by  one  with  a  straight  platinum  wire,  sows  a  separate  tube  of  broth 
with  each  colony,  and  then  smears  the  wire  in  a  straight  line  across  a  cover-glass. 
The  first  colony  is  smeared  along  one  edge  of  the  cover-glass,  the  others  at  right 
angles  to  it.  In  this  way  not  only  is  it  possible  to  make  4  to  9  separate  preparations 
on  one  cover-glass  from  the  different  colonies  of  a  single  culture-tube,  but  pure  cul- 
tures of  each  colony  also  are  available  for  further  examination.  ] 

A  diagnosis  of  diphtheria  [infection]  must  be  given  in  all  cases  in  which 
colonies  consisting  of  organisms  having  the  morphological  appearance  and 
giving  the  staining  reactions  of  the  diphtheria  bacillus  are  found  on  the 
serum  sown  with  the  suspected  material.  [In  cases  of  faucial  diphtheria 
such  colonies  will  generally  be  present  in  large  numbers  ;  in  laryngeal 
diphtheria  they  are  sometimes  few  in  number — this  is  often  the  case  also  with 
convalescents  and  "  contacts."] 

C.  Inoculations. — When  absolute  confirmation  of  the  positive  microscopical 
and  cultural  results  is  desired  resort  must  be  had  to  animal  inoculation. 
Several  of  the  suspected  diphtheria  colonies  must  be  inoculated,  because 
bacilli  of  different  degrees  of  virulence  may  be  present  in  the  same  membrane. 

Each  colony  is  dealt  with  as  follows  :  Sow  a  portion  of  a  colony  in  broth 
(taking  every  care  that  the  needle  touches  no  other  colony),  incubate  for  24 
hours,  and  after  verifying  the  purity  of  the  culture  inoculate  1  c.c.  sub- 
cutaneously  into  an  adult  guinea-pig  :  the  animal  dies  more  or  less  rapidly 
according  to  the  virulence  of  the  organism  (p.  247).  Should  the  organism 
prove  non- virulent  for  the  guinea-pig  test  it  on  a  small  bird  in  a  similar 
manner. 


BACTERIOLOGICAL  DIAGNOSIS  273 


[3.  Summary  of  diagnostic  tests.] 

[As  some  difficulty  may  be  experienced  in  differentiating  the  diphtheria 
bacillus  from  other  organisms — and  especially  from  Hofmann's  bacillus — it 
will  be  convenient  to  summarize  the  purely  specific  characteristics  of  the 
organism  :  references  are  given  to  the  pages  on  which  these  characteristics 
are  discussed  in  detail  and  also  to  the  pages  on  which  the  reactions  under 
similar  circumstances  of  Hofmann's  bacillus  are  considered  :  the  reader  will 
thus  be  in  a  position  readily  to  form  a  diagnosis. 

The  true  diphtheria  bacillus  is  characterized  by  its  : 

(a)  Macroscopic  growth  on  serum  and  morphology  (p.  250  diphtheria  bacillus : 
and  p.  273  Hofmann's  bacillus). 

(/3)  Power  of  producing  acid  in  glucose  broth  (pp.  256  and  274). 

(y)  Invisible  growth  on  potato  (p.  254). 

(8)  The  lesions  produced  in  inoculated  guinea-pigs  (pp.  247,  255  and  274). 

Bacillus  pseudo-diphtherice. 

(Hofmann's  bacillus.) 

In  the  mouths  of  healthy  persons  and  in  some  cases  of  non-diphtheritic 
sore  throat  a  non-virulent  bacillus  described  by  Loeffler  as  the  pseudo- 
diphtheria  bacillus  is,  as  has  already  been  indicated,  not  infrequently  present. 

[According  to  Graham-Smith,  Hofmann's  bacillus  is  most  commonly  found 
in  the  throats  of  the  poorer  classes,  especially  the  scholars  in  the  public 
schools  (51  per  cent,  to  56  per  cent.).  The  children  attending  better-class 
schools  are  less  commonly  found  to  harbour  this  bacillus  in  their  throats 
(8  per  cent,  and  15  per  cent.).  In  adults  the  extent  of  infection  is  less  than 
in  children,  but  it  is  greater  amongst  the  poor  (20  per  cent.)  than  amongst 
the  well-to-do  (9  per  cent.).] 

This  organism  is  by  some  observers  (Loeffler,  Hofmann,  [Cobbett]  and  others) 
sharply  differentiated  from  the  diphtheria  bacillus,  while  others  (Roux  and 
Yersin)  have  brought  forward  arguments  in  favour  of  its  identity  with  the 
diphtheria  bacillus  :  in  the  view  of  the  latter  school  the  pseudo-diphtheria 
bacillus  is  merely  a  diphtheria  bacillus 
devoid  of  virulence  (see  also  pp.  274  and  275). 

[Morphology. — When  taken  from  young 
serum  or  alkalized  glucose  serum  cultures, 
stained  with  a  weak  solution  of  Loeffler's 
blue  and  mounted  in  the  stain,  the  bacillus 
of  Hofmann  exhibits  great  uniformity  of 
type  ;  it  is  oval,  stains  deeply,  has  no  gran- 
ules, but  shows  one  unstained  septum.  The 
arrangement  too  is  quite  different  from  that 
of  the  diphtheria  bacillus:  Hofmann's 
bacillus  ranges  itself  in  parallel  groups 
resembling  a  paling.  Occasionally  the  or- 
ganism departs  from  this  typical  form,  and 
numerous  many-banded  forms  O3cur  ;  on 
sub-culture  however  these  many-banded 
forms  revert  to  the  type  already  described.  Oc.  4,  obj.  Tuh,  Zeiss. 

[Staining  reactions. — Stained  with  a  weak 

solution  of  Loeffler's  blue  and  mounted  in  the  stain,  the  organism  will  be 
found  to  be  a  deeply  and  uniformly  stained  oval  bacillus  with  one  unstained 
septum.  On  running  a  drop  of  acetic  acid  (5  per  cent.)  under  the  cover-glass 

s 


274  HOFMANN'S   BACILLUS 

the  whole  organism  decolourizes.  (Occasionally  a  minute  granule  may  be  seen 
at  the  poles  of  some  of  the  bacilli,  but  these  minute  specks  present  a  very 
different  appearance  to  the  granules  seen  in  the  diphtheria  bacillus,  and  are 
relatively  few  in  number.)  If  stained  with  Neisser's  blue,  washed  in  water 
and  counterstained  with  Bismarck  brown  (1  minute  in  each  stain)  the  bacilli 
will  be  stained  uniformly  brown  ;  blue  granules  will  be  absent  or  very 
indistinct. 

[The  organism  like  the  diphtheria  bacillus  retains  the  violet  by  Gram's 
method. 

[Cultural  characteristics.— On  serum  or  alkalized  glucose  serum  the  growth 
is  more  rapid  than  that  of  the  diphtheria  bacillus,  and  the  colonies  are  larger 
and  whiter  and  do  not  take  up  the  pigment  in  the  serum. 

[Bio-chemical  reactions. — When  grown  for  48  hours  in  a  nutrient  broth 
neutral  to  litmus  and  containing  1  per  cent,  glucose  no  acid  is  formed  ;  on 
the  contrary  a  slight  increase  in  alkalinity  takes  place.] 

Rothe  recommends  the  following  medium.— 

Neutral  broth,.  1  part. 

Ox  serum,         -  4  parts. 

10  per  cent,  glucose  litmus  solution,    -  £  part. 

Solidify. 

The  diphtheria  bacillus  on  the  other  hand  turns  this  medium  red. 

[Virulence. — A  guinea-pig  inoculated  with  2  c.c.  or  more  of  a  48-hour 
culture  in  broth  remains  perfectly  well,  not  even  a  local  oedema  resulting. 

[Immunity. — Hofmann's  bacillus  produces  no  substances  toxic  to  laboratory 
animals.  Petrie  experimenting  with  Hofmann's  bacillus  finds  that  "  no 
substances  capable  of  neutralizing  diphtheria  antitoxin  are  present  in  the 
nitrates  of  the  pseudo-diphtheria  bacillus";  and  his  attempts  to  immunize 
horses  with  this  bacillus  against  diphtheria  toxin  were  negative.] 

The  relation  of  Hofmann's  bacillus  to  the  diphtheria  bacillus.— [Petrie's 
experiments  on  immunity  accentuate-  the  differences  between  the  two 
organisms — which  have  been  detailed  above — and  diminish  the  probability 
that  they  stand  in  close  relation  to  each  other. 

[Further  support  of  the  latter  view  is  afforded  by  the  fact  that  no  satis- 
factory evidence  of  the  conversion  of  the  one  organism  into  the  other  has  yet 
been  brought  forward,  though  numerous  experiments  have  been  conducted 
for  that  purpose. 

[The  non-identity  of  the  diphtheria  bacillus  with  Hofmann's  bacillus 
receives  further  confirmation  from  certain  practical  observations.  In  the 
first  place,  as  has  already  been  shown,  the  distribution  of  the  diphtheria 
bacillus  is  limited  to  the  throats  of  those  who  have  been  in  contact  with  cases 
of  diphtheria  or  with  diphtheria-infected  contacts,  while  Hofmann's  bacillus 
is  a  widely  distributed  organism  present  in  the  throats  of  a  considerable  pro- 
portion of  the  ordinary  healthy  population.  Secondly,  Cobbett  successfully 
stamped  out  two  epidemics  of  diphtheria,  one  at  Colchester  and  one  at 
Cambridge,  by  isolating  only  those  in  whose  throats  diphtheria  bacilli  were 
found,  those  who  harboured  the  bacillus  of  Hofmann  and  no  diphtheria 
bacilli  being  treated  as  non-infected  individuals.  Other  observers  have  had 
similar  experiences. 

[The  above  then  is  the  case  of  those  who  regard  the  Klebs-Lceffler  and 
Hofmann's  bacillus  as  distinct  species. 

[The  following  arguments,  which  are  advanced  in  favour  of  the  identity  of  the 
two  organisms,  may  be  prefaced  by  a  quotation  from  Graham-Smith  supporting 
an  earlier  statement  to  the  same  effect  by  Cobbett.  "  The  authority  of  Roux  (1890) 
whose  opinion  justly  carries  great  weight  has  often  been  quoted  in  support  of  the 


HOFMANN'S  BACILLUS  275 

idea  that  Hofmann's  bacillus  is  related  to  the  diphtheria  bacillus.  But  this  is  not 
right,  for  his  remarks  on  the  pseudo-diphtheria  bacillus  were  made  in  comparatively 
early  days,  when  the  importance  of  acid  production  had  not  been  generally  recog- 
nized and  before  Hofmann's  bacillus  had  been  clearly  distinguished  from  the  so-called 
non- virulent  diphtheria  bacillus."] 

A.  From  the  morphological  point  of  view  the  differences  between  the  two  organisms 
are  insignificant.     The  pseudo-diphtheria  bacillus  is  as  a  rule  shorter  than  the 
diphtheria  bacillus  but  Martin  has  shown  that  this  is  not  always  the  case  :  the  length 
of  the  diphtheria  bacillus  is  subject  to  great  variation  and  the  variations  in  length 
afford  in  some  degree  an  index  of  its  virulence. 

Too  great  importance  must  not  be  attached  to  certain  characteristics  said  to  be 
possessed  by  the  pseudo-diphtheria  bacillus  by  those  who  consider  it  a  distinct 
species,  and  the  constancy  of  which  have  not  been  confirmed.  Deguy,  for  instance, 
says  that  the  pseudo-diphtheria  bacillus  is  motile  (?) ;  according  to  Barbier  it 
shows  no  granules  and  stains  more  deeply  than  the  diphtheria  bacillus  by  Gram's 
method,  etc.  Characteristics  based  upon  the  staining  of  the  metachromatic  granules 
are  of  no  value  whatever  for  purposes  of  identification  (p.  252). 

B.  The  pseudo-diphtheria  bacillus  occasionally  produces  an  oedema  at  the  site  of 
inoculation  when  inoculated  into  guinea-pigs  but  never  leads  to  a  fatal  result.     The 
same  organism  may  kill  small  birds.     Roux  and  Yersin  were  able  to  increase  the 
virulence  of  an  organism  which  produced  a  local  oedema  in  guinea-pigs  by  mixing 
it  with  a  streptococcus.     The  virulence  is  therefore  not  a  fixed  quantity. 

C.  Objection  to  the  identity  of  the  two  bacilli  may  be  taken  on  the  ground  that  a 
totally  non- virulent  pseudo-diphtheria  bacillus  has  so  far  never  been  made  virulent. 
The  objection,  however,  cannot  be  upheld  in  view  of  an  experiment  of  Roux  and 
Yersin :    virulent  bacilli  were  isolated  from  the  throat  of  a  person  suffering  from 
diphtheria ;  as  the  patient  progressed  to  convalescence,  the  bacilli  became  less  viru- 
lent and  were  finally  totally  avirulent  and  their  virulence  could  not  be  restored. 

Martin  has  proved  that  some  short  bacilli  which  are  not  fatal  to  guinea-pigs  are 
degenerated  diphtheria  bacilli. 

Martin's  experiment. — An  eight  months  old  broth  culture  of  a  long,  very  virulent 
bacillus  was  subcultivated  in  broth ;  the  organism  failed  to  grow,  but  when  sown 
on  agar  covered  with  a  film  of  recently  prepared  veal  broth  gave  a  growth  of  a  short 
bacillus  which  remained  short  in  subsequent  subcultivations.  On  inoculation,  it 
did  not  kill  guinea-pigs  but  gave  rise  to  a  local  oedema  from  which  the  short  bacillus 
was  recovered. 

This  artificially  obtained  short  bacillus  was  pathogenic  for  sparrows,  and  on 
inoculating  a  normal  sparrow  and  a  sparrow  which  had  been  treated  with  anti- 
diphtheria  serum  each  with  O'l  c.c.  of  a  broth  culture  of  the  organism,  the  normal 
sparrow  died  while  the  other  survived.  This  short  bacillus,  non- virulent  for  guinea- 
pigs,  was  therefore  undoubtedly  a  diphtheria  bacillus. 

D. — In  suitable  culture  media,  non- virulent  diphtheria  bacilli  frequently  produce 
toxin  neutralizable  by  antidiphtheria  serum  (Martin). 

[In  this  case  apparently  a  non- virulent  diphtheria  bacillus  was  under  observation. 
Such  bacilli  admittedly  exist.  See  p.  255.] 

E. — According  to  Spronck,  a  prophylactic  inoculation  of  antidiphtheria  serum 
prevents  the  development  of  a  local  oedema  when  a  diphtheria  bacillus  is  inoculated 
but  not  when  a  pseudo-diphtheria  bacillus  is  inoculated.  This  however  does  not 
appear  to  be  a  constant  phenomenon. 


CHAPTER  XVI. 
BACILLUS  PYOCYANEUS. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  276. 

Section  II.— Morphology,  p.  277. 

1.  Microscopical  appearance  and  staining  reactions,  p.  277.     2.  Cultural  charac- 
teristics, p.  278. 
Section  III. — Biological  properties,  p.  279. 

1.  Pigments,  p.  279.     2.  Toxins,  p.  280.     3.  Vaccination  and  serum  therapy,  p.  280. 
4.  Agglutination,  p.  281.     5.  Antagonism,  p.  281. 
Section  IV. — Detection  and  isolation  of  the  organism,  p.  281. 

THE  cause  of  blue  suppuration  was  discovered  by  Gessard. 

Blue  pus  is  rarely  seen  nowadays  though  it  was  very  common  before  the 
introduction  of  antiseptics.  The  bacillus  pyocyaneus  is  always  associated 
in  these  conditions  with  the  ordinary  micro-organisms  of  suppuration ;  its 
presence  in  a  wound  is  simply  a  complication  and  that  not  of  a  serious 
nature. 

The  bacillus  pyocyaneus  may  invade  the  tissues  of  the  body  when  the 
resistance  of  the  latter  has  been  broken  down  by  some  pre-existing  patho- 
logical condition.  It  has,  for  instance,  often  been  found  in  the  internal 
organs  in  cases  of  enteric  fever  ;  Calmette  found  it  in  the  blood  of  persons 
suffering  from  chronic  dysentery  [and  Williams  and  Lartigan  in  association 
with  diarrhoea].  [Of  twenty- three  cases  of  pyocyaneus  infection  occurring 
in  British  Guiana  and  recorded  by  Minett  and  Duncan  six  were  cases  of 
acute  filariasis  and  six  others  showed  intestinal  ulceration.]  On  the  other 
hand  it  may  be  the  primary  cause  of  disease  :  e.g.  of  enteritis  (Legros),  appen- 
dicitis (Coyne  and  Hobbs),  otitis,  pseudo-membranous  sore  throat  (Calvo 
Ignacig),  etc.  :  some  twenty  cases  of  a  generalized  infection  with  the  bacillus 
pyocyaneus  have  moreover  been  recorded. 

[Dogs  are  liable  to  infection  with  the  bacillus  pyocyaneus  and  in  these  animals 
the  symptoms  may  clinically  resemble  rabies ;  and  moreover  the  inoculation  of 
brain  tissue  from  the  affected  animals  into  normal  rabbits  and  guinea-pigs  produces 
symptoms  similar  to  those  seen  in  the  original  animal.] 

The  organism  is  sometimes  found  in  the  soil,  in  dust  and  in  water.  Besson  has 
recorded  its  almost  constant  presence  in  the  waters  of  the  regency  of  Tunis  where 
blue  suppuration  is  very  common  and  where  serious  infections  are  often  com- 
plicated by  the  bacillus  pyocyaneus. 

SECTION  I.— EXPERIMENTAL   INOCULATION. 

The  bacillus  pyocyaneus  is  pathogenic  to  rabbits,  guinea-pigs,  rats  and 
mice. 


MORPHOLOGY  277 

Rabbits. — Sub-cutaneous  inoculation  in  rabbits  is  rarely  fatal :  intra- 
peritoneal  inoculation  does  not  give  constant  results. 

The  injection  of  1  c.c.  of  a  broth  culture  into  the  ear  vein  of  a  rabbit,  which 
is  the  most  certain  method  of  producing  infection,  causes  an  acute  disease. 
The  animal  suffers  from  fever,  albuminuria  and  diarrhoea  and  dies  in  24-48 
hours.  The  organisms  are  very  numerous  in  the  liver,  spleen  and  kidneys  but 
only  a  few  are  to  be  found  in  the  blood. 

Inoculation  of  smaller  doses  of  culture  leads  to  a  chronic  but  not  necessarily 
fatal  disease  in  the  rabbit  which  is  characterized  by  wasting,  paralysis 
of  the  limbs  and  convulsions.  If  death  take  place  it  is  not  uncommon  to 
find  post  mortem  a  true  nephritis  with  small  contracted  kidney,  and  even 
hypertrophy  of  the  left  ventricle  of  the  heart.  In  some  cases  there  is  amyloid 
degeneration  of  the  kidneys  and  infarcts  may  be  present  in  the  alimentary 
mucosa  (Charrin). 

By  feeding  rabbits  with  the  organism  Brau  produced  fatty  degenera- 
tion of  the  liver  and  ulceration  of  the  intestine  followed  by  generalization  of 
the  bacillus  in  the  tissues  of  the  animal. 

Guinea-pigs.— In  guinea-pigs,  sub-cutaneous  inoculation  produces  a  local 
swelling  followed  by  ulceration  :  the  organism  then  becomes  disseminated 
and  the  animal  dies.  Intra-peritoneal  inoculation  is  more  severe  and  death 
takes  place  rapidly  ;  the  bacillus  is  found  in  the  blood. 

Rats.  Mice. — The  results  are  the  same  as  in  the  guinea-pig  :  mice  are 
very  susceptible. 

An  organism  isolated  by  Besson  from  the  water  of  Zaghouan  killed  white 
rats  in  20-36  hours  when  inoculated  sub-cutaneously  in  doses  of  O20  c.c. 
Post  mortem  the  abdominal  organs  *were  congested,  the  peritoneum  contained 
a  small  quantity  of  an  almost  clear  fluid  and  the  intestines  showed  an  early 
stage  of  ulceration  in  many  of  the  Peyer's  patches  :  in  two  cases  the  animals 
had  heematuria  and  the  organism  was  found  in  the  blood,  liver,  kidneys, 
peritoneal  fluid,  intestinal  contents,  and  in  the  urine  in  the  bladder. 

Increase  of  virulence. — The  virulence  of  the  bacillus  pyocyaneus  can  be 
increased  by  passage  through  rabbits  to  such  an  extent  that  after  a  few 
passages  a  dose  of  0*1  c.c.  of  a   broth   culture 
rapidly  kills  animals  of  this  species.  £V 

SECTION  II.— MORPHOLOGY.  ^  ^      v    ^J      ^ 

1.  Microscopical  appearance.  ,M    /,Jn         /'  * 

The  bacillus  pyocyaneus  is  a  small  motile  rod-  •       vV      ^% 

shaped  [non-spore  bearing]  organism  with  rounded          //       f       ' 
ends,  and  of  variable  size.     Its  average  length  is  "**   x 

about  1-5/A  and  its  breadth  O5  to  0'6/x.     It  has         /  «*    * 

one  flagellum  situated  terminally  [monotrichous].         ^      v^       ft   % 

Staining  reactions.— The  bacillus  pyocyaneus  is  Fm  17^_Bacmus  pyocyaneus. 
easily  stained  by  the  basic  aniline  dyes  and  is  Film  from  an  agar  culture  (dilute 
gram-positive.  ^S^^'  (OcJii'obj"l=th' 

The  bacillus  stains  rather  badly  by  Gram's  method : 

some  strains  stain  feebly  and  irregularly,  and  decolourization  takes  place  easily  if 
the  action  of  the  alcohol  be  prolonged. 

Morphological  variations. — The  morphology  of  the  bacillus  pyocyaneus 
undergoes  considerable  change  if  sown  in  media  containing  small  amounts 
of  antiseptics  (Guignard  and  Charrin).  For  instance,  in  broth  containing 
O02  per  cent,  of  carbolic  acid  the  organism  is  long  and  filamentous.  The 
addition  of  alcohol  and  bichromate  of  potassium  to  the  medium  have  a 


278 


THE  BACILLUS   OF  BLUE  PUS 


similar  effect.     In  broth  containing  boric  acid  the  bacillus  assumes  a  spiral 
form,  and  in  media  containing  creosote  it  looks  like  a  coccus  (figs.  174  to  177). 


FIG.  174.— Culture  in  4  per  cent, 
alcohol  broth. 


FIG.  175. — Culture  in  0'015  per  cent, 
potassium  bichromate  broth. 


FIG.  176. — Culture  in  0'70  per  cent, 
boric  acid  broth. 


FIG.  177.  —  Culture  in  O'lO  per  cent. 
creosote  broth. 


FIGS.  174-177.  —  Different  morphological  appearances  presented  by  the  Bacillus 
pyocyaneus  when  grown  in  broth  containing  traces  of  antiseptics  (after  Guignard 
and  Charrin). 


2.  Cultural  characteristics. 

Conditions  of  growth. — The  bacillus  pyocyaneus  is  a  facultative  aerobe  but 
the  pigment  is  only  formed  in  presence  of  air.  It  grows  at  all  temperatures 
between  15°  and  43°  C.,  the  optimum  temperature  being  about  35°-37°  C. 


FIG.  178.  —  Bacillus 
pyocyaneus — broth  cul- 
ture— 1st  day. 


FIG.  179.  —  Bacillus 
pyocyaneus — broth  cul- 
ture— 3rd  day. 


FIG.  180.  —  Bacillus 
pyocyaneus — broth  cul- 
ture— 7th  day. 


Characters  of  growth  on  various  media,  (i)  Broth.— After  incubating  at 
37°  C.  for  8  hours  the  medium  becomes  cloudy,  and  then  a  greenish 
fluorescence  appears  at  first  limited  to  the  upper  part  of  the  medium  then 


ext 


BIOLOGICAL  PROPERTIES  279 


extending  throughout.  During  the  next  few  days  a  white  wrinkled  pellicle 
forms  on  the  surface,  which  as  growth  proceeds  becomes  thicker,  dry  and 
brown  and  falls  to  the  bottom  of  the  tube  where  it  forms  a  dirty  white 
deposit,  the  broth  at  the  same  time  becoming  deep  green 
in  colour  and  afterwards  brownish.  The  culture  is  viscous 
and  ropy  and  has  a  peculiar  odour. 

(ii)  Gelatin.  Stab  culture. — After  incubating  for  2  days 
at  20°  C.  small  colonies  appear  along  the  line  of  the  stab  : 
these  coalesce  and  form  a  white  streak :  liquefaction 
commences  about  the  third  day  (champagne  glass  lique- 
faction) and  rapidly  extends  to  the  walls  of  the  tube.  The 
medium  is  coloured  green. 

Isolated  colonies. — Small,  yellowish,  granular  colonies 
appear  on  the  plates  after  incubating  for  2  days.  Lique- 
faction occurs  round  them  and  gradually  extends  through- 
out the  plate.  The  gelatin  assumes  a  green  tint. 

(iii)  Agar. — After  incubating  for  24  hours  at  37°  C. 
a  greenish  streak  appears  on  the  agar  which  rapidly 
spreads  over  the  surface,  the  agar  taking  a  fluorescent 
green  colour. 

(iv)  Potato. — Along  the  line  of  sowing  a  thick  brown 
layer  is  formed,  and  if  this  be  removed  the  surface  of 
the  potato  beneath  becomes  green  on  exposure  to  air. 

SECTION   III.— BIOLOGICAL   PROPERTIES. 

1.   Pigments  (Gessard).  FIQ 

When  a  broth    culture    of  the   bacillus  pyocyaneus  is   cuiture^on  agar-3  days 
shaken  up  with  a  little  chloroform  and  allowed  to  stand 
for  a  moment  the  chloroform  separates  at  the  bottom  of  the  tube  and  is 
coloured  pure  blue,  while  a  beautiful  fluorescent  green  watery  liquid  floats 
to  the  surface. 

The  bacillus  pyocyaneus  secretes  three  pigments,  one  blue  (pyocyanine) ; 
another  fluorescent  and  green  and  identical  with  the  pigment  produced  by 
saprophytic  fluorescent  bacilli ;  the  third  is  greenish  and  non-fluorescent 
and  of  little  importance. 

In  contact  with  air  pyocyanine  oxydizes  and  forms  a  brown  substance, 
pyoxanthose. 

Pyocyanine  is  easily  obtained  by  extracting  a  broth  or  agar  culture  with  chloro- 
form. In  the  case  of  agar  it  is  only  necessary  to  leave  the  chloroform  on  the  culture 
for  a  few  hours  without  shaking.  The  chloroform  acquires  a  blue  colour,  and  if 
evaporated  long  blue  needles  of  pyocyanine  crystallize  out.  Solutions  of  pyo- 
cyanine are  turned  red  by  dilute  acids  but  the  blue  colour  is  restored  on  the  addition 
of  an  alkali.  Cultures  in  broth  or  peptone  solution  retain  their  colour  after  filtration 
through  a  Chamberland  bougie.  Pyocyanine  is  not  toxic. 

The  formation  of  pyocyanine  and  of  the  green  pigment  may  be  varied 
at  will  and  even  suppressed  by  growing  the  organism  on  different  culture 
media. 

In  a  solution  of  peptone,  Gessard  was  able  to  suppress  the  formation  of  the  green 
pigment  and  the  culture  then  had  a  very  pretty  blue  colour  (this  phenomenon  cannot 
be  obtained  with  all  peptones).  In  the  same  way,  on  glycerin-peptone-agar  (the 
test  medium  of  Gessard)  the  amount  of  pyocyanine  produced  is  considerably  increased. 
Pyocanine  is  the  only  pigment  formed  when  the  organism  is  sown  in  a  10  per  cent, 
gelatin  medium  containing  a  little  glycerin  and  incubated  at  35°  C. 

On  the  other  hand,  the  green  pigment  is  formed  to  the  exclusion  of  the  others  when 


280  THE   BACILLUS   OF  BLUE   PUS 

a  medium  containing  2  per  cent,  glucose  is  used,  and  the  same  result  is  obtained 
with  white  of  egg. 

No  pigment  at  all  is  formed  in  broth  containing  5-6  per  cent,  of  glucose,  or  when 
the  bacillus  is  grown  on  the  serum  of  immunized  animals. 

Gessard  was  successful  in  producing  strains  of  the  bacillus  some  of  which 
secreted  the  green  pigment,  and  others  pyocyanine.  Wasserzug  grew  the 
bacillus  on  slightly  acid  media  and  found  that  it  had  altogether  lost  its 
power  of  pigment  production.  Charrin  obtained  a  similar  result  by  sub- 
culturing  in  broth  and  incubating  the  cultures  at  42°  C. 

Melanogenie  variety. — Cassin  and  Gessard  studied  a  strain  of  the  bacillus  pyo- 
cyaneus which  when  sown  in  broth  produced  in  the  first  instance  the  ordinary  pigment 
but  later  a  dark  brown  and  finally  a  black  pigment.  Cultures  on  potato  formed  a 
deep  brown  layer  which  soon  turned  black.  This  production  of  black  pigment  was 
found  to  be  possible  only  when  tyrosin  was  present  in  the  media.  In  a  "  mineral  " 
medium  such  as  the  following : — 

Ammonium  succinate,        -  1      gram 


Sodium  phosphate,   - 
Magnesium  sulphate, 
Calcium  chloride  (crystals), 
Water,     .... 


1 

2 '5  grams. 
1-25 


1000 


this  bacillus  produces  no  black  pigment,  the  growth  having  all  the  characteristics 
of  an  ordinary  bacillus  pyocyaneus  ;  but  by  adding  0'5  per  cent,  of  tyrosin  to  the 
medium  a  rose  colour  is  at  first  produced  which  later  becomes  a  deep  brown. 

2.  Toxins. 

Filtered  cultures  of  the  bacillus  pyocyaneus  inoculated  in  sufficient  quantity 
into  rabbits  either  cause  death  with  all  the  symptoms  of  the  acute  experi- 
mental disease  or  lead  to  cachexia  and  paralysis  which  may  also  terminate 
fatally. 

Wassermann  obtained  a  very  toxic  product  by  incubating  broth  cultures 
for  40  days  and  then  sterilizing  them  by  leaving  them  to  stand  under  toluol 
for  a  week.  These  cultures  killed  guinea-pigs  in  doses  of  0*5  c.c.  when 
inoculated  intra-peritoneally. 

The  toxicity  of  the  cultures  is  not  due  to  pyocyanine  but  to  certain  other 
substances,  some  of  which  are  volatile,  easily  destroyed  and  have  merely  a 
transitory  action,  while  others  are  non-volatile.  The  non- volatile  products 
may  be  divided  into  two  groups  ;  those  of  the  first  group — the  most  toxic — 
are  precipitated  by  alcohol,  the  others  are  soluble  in  alcohol  (Arnaud  and 
Charrin). 

If  injected  into  the  veins  of  a  rabbit  the  products  of  the  bacillus  pyocyaneus 
rapidly  lead  to  the  death  of  the  animal  without  an  incubation  period.  This  absence 
of  an  incubation  period  is  to  be  referred  chiefly  to  the  action  of  the  volatile  con- 
stituents which  are  not  a  part  of  the  true  toxins. 

Pyocyanolysin.— Cultures  in  neutral  peptone-broth,  7-30  days  old,  filtered 
or  killed  by  toluol  or  heat  (15  minutes  at  60°  C.),  have  a  powerful  hsemolytic 
action  on  freshly  denbrinated  ox,  sheep  and  rabbit  blood  (Bulloch  and  Hunter). 

Cultures  3-4  weeks  old  are  the  best  for  demonstrating  these  properties. 
The  cultures  are  strongly  alkaline  in  reaction. 

Pyocyanolysin  withstands  high  temperatures.  The  hsemolytic  property 
is  not  destroyed  by  heating  cultures  at  100°  C.  for  15  minutes,  and  it  is  also 
said  to  be  unchanged  by  heating  at  120°  C.  for  30  minutes  (Weingeroff,  and 
Breymann). 

3.  Vaccination— Serum  therapy. 

If  an  average  dose  of  a  culture  be  inoculated  beneath  the  skin  of  a  rabbit 
the  animal  suffers  no  harm.  Rabbits  can  be  immunized  by  inoculating  them 


in  t 


ISOLATION  OF  THE   ORGANISM  281 


in  this  manner  with  doses  of  O'5-l  c.c.  of  a  broth  culture  on  five  or  six  occa- 
sions at  intervals  of  3  or  4  days  :  or  by  inoculating  them  with  small  doses 
of  filtered  cultures  or  cultures  heated  at  115°  C. 

The  blood  and  urine  of  animals  treated  with  filtered  cultures  will  also 
immunize  animals. 

Wassermann  immunized  guinea-pigs  by  inoculating  them  in  the  peritoneal 
cavity  with  gradually  increasing  doses  of  living  culture  or  toxin.  Guinea- 
pigs  vaccinated  with  living  cultures  show  a  permanent  immunity  to  the 
organism,  but  an  inoculation  of  toxin  is  fatal  to  them.  Their  serum  is  pro- 
phylactic and  has  feeble  therapeutic  properties,  but  is  not  antitoxic.  Guinea- 
pigs  vaccinated  with  toxin  are  immune  against  both  the  organism  and 
the  toxin,  and  their  serum  is  prophylactic,  distinctly  therapeutic  and 
antitoxic. 

The  bacillus  pyocyaneus  will  grow  in  the  serum  obtained  from  immunized  animals  : 
it  preserves  its  shape,  its  vitality  and  its  virulence,  but  forms  agglutinated  colonies 
(Charrin  and  Roger,  Gheorghiewsky)  and  produces  no  pigment.  The  serum  of 
immunized  animals  is  therefore  not  bactericidal ;  it  is  simply  agglutinating  in  vitro. 
The  agglutinating  property  does  not  run  parallel  with  the  prophylactic  property. 

4.  Agglutination. 

The  agglutinating  property  of  the  blood  of  vaccinated  animals  has  just 
been  referred  to.  The  blood  of  infected  persons  when  diluted  1  in  40  to  1  in 
100  similarly  agglutinates  the  bacillus,  and  normal  human  serum  has  in  some 
cases  an  agglutinating  action  in  a  dilution  of  1  in  10.  To  demonstrate  the 
agglutination  it  is  best  to  add  the  serum  to  an  emulsion  prepared  by  diluting 
in  normal  saline  solution  the  centrifuged  deposit  of  broth  cultures  at  least 
24  hours  old. 

5.  Antagonism. 

The  Bacillus  pyocyaneus  impedes  the  growth  of  anthrax  in  cultures.  In 
the  same  way  by  inoculating  a  mixture  of  the  anthrax  bacillus  and  the 
bacillus  pyocyaneus  into  animals  susceptible  to  anthrax  the  animals  do  not 
become  infected  with  anthrax.  Porcelain-filtered  cultures  of  the  bacillus 
pyocyaneus  possess  the  same  properties  (Blagovetschensky). 

Similarly  the  bacillus  pyocyaneus  will  prevent  the  development  of  the 
cholera  vibrio  (Kitasato). 

Rumpf  has  recorded  a  parallel  antagonism  between  the  bacillus  pyocyaneus  and 
the  typhoid  bacillus  ;  he  successfully  treated  65  cases  of  enteric  fever  by  inoculating 
the  patients  with  sterilized  cultures  of  the  bacillus  pyocyaneus.  It  does  not  appear 
that  much  faith  should  be  put  in  these  statements.  Analogous  investigations 
undertaken  on  the  guinea-pig  by  Besson  led  him  to  the  conclusion  that  these  animals 
when  treated  with  filtered  cultures  of  the  bacillus  pyocyaneus  are  more  than  normally 
susceptible  to  the  action  of  the  typhoid  and  colon  bacilli :  moreover,  infection 
with  the  bacillus  pyocyaneus  has  been  recorded  coincidently  with  a  fatal  attack 
of  enteric  fever  in  man. 


SECTION   IV.— DETECTION,   ISOLATION  AND   IDENTIFICATION  OF 
THE   BACILLUS   PYOCYANEUS. 

The  presence  of  the  bacillus  pyocyaneus  in  pus  is  detected  by  the  blue 
colour  of  the  dressings  and  by  the  characteristic  smell  of  the  wound. 

Pus  should  be  examined  by  staining  films  with  gentian-violet  or  thionin. 
The  bacilli  can  be  easily  isolated  on  gelatin  plates  on  which  they  produce  a 
characteristic  appearance.  At  the  post  mortem  examination  cultures  should 


282  THE   BACILLUS   OF  BLUE  PUS 

also  be  sown  in  broth  with  blood  and  scrapings  of  tissues.  The  characteristic 
colour  produced  by  the  bacillus  is  sometimes  only  apparent  after  several  sub- 
cultures in  broth  or  on  agar. 

Inoculations  should  be  made  into  the  peritoneal  cavity  of  a  guinea-pig 
or  into  the  ear  vein  of  a  rabbit. 

Besson  isolated  the  organism  from  water  by  sowing  in  MetchnikofFs  gelatin  - 
peptone-salt  medium  (p.  33).  When  growth  appeared  after  12—15  hours  a  sub- 
culture was  made  in  the  same  medium,  and  a  trace  of  this  second  culture  was  sown  on 
gelatin  plates  from  which  the  organism  was  easily  obtained  in  pure  culture. 


CHAPTER  XVII. 
THE   BACILLUS   OF  SWINE   ERYSIPELAS.1 

Introduction. 

Section  I. — Experimental  inoculation,  p.  284. 
Section  II. — Morphology  and  cultural  characteristics,  p.  284. 
Section  III. — Biological  properties,  p.  286. 

Section  IV. — The  detection,  isolation  and  identification  of  the  bacillus,  p.  287. 
The  bacillus  of  mouse  septicaemia  (Bacterium  murisepticum),  p.  288. 

SWINE  erysipelas  (measles)  is  due  to  a  bacillus  discovered  by  Pasteur  and 
Thuillier,  and  of  which  the  classical  description  was  given  by  Loaffler. 

A  very  large  number  of  deaths  among  swine  are  attributable  to  swine  erysipelas 
and  the  disease  becomes  therefore  of  considerable  economic  importance.  The 
acute  form  of  the  disease  is  nearly  always  fatal,  and  in  infected  herds  about  50  per 
cent,  of  the  animals  die. 

Swine  are  liable  to  the  disease  between  the  ages  of  6  months  and  2  years ;  under 
3  months  old  they  are  immune  and  beasts  more  than  2  years  old  are  rarely  infected. 

Highly  bred  swine,  such  as  the  English  breeds,  are  the  most  susceptible,  while 
wild  animals  are  immune. 

Swine  become  infected  by  feeding  upon  the  excreta  of  infected  animals  ;  they 
are  almost  the  only  animals  susceptible  to  the  spontaneous  disease ;  pigeons,  [mice], 
and  rabbits  which  have  frequented  infected  pig- sties  are  however  sometimes  attacked. 

The  flesh  of  suspected  animals,  and  even  of  those  dead  of  the  disease,  is  frequently 
consumed  as  food  without  apparently  causing  any  harmful  effects  in  man :  but 
cases  of  painful  erythema  have  been  noticed  following  the  accidental  inoculation 
of  the  virus. 

Swine  erysipelas  occurs  in  two  forms,  acute  and  chronic. 

The  acute  form  of  the  disease  is  characterized  by  the  appearance  of  bright  or 
dark  red  purpuric  spots  on  the  skin,  chiefly  about  the  ears,  the  anus  and  vulva,  the 
internal  surface  of  the  thighs  and  the  groins.  The  animal  suffers  from  diarrhoea  : 
it  grunts  dismally  and  remains  lying  down  hidden  in  its  bedding  with  its  tail  uncurled 
and  hanging  down  :  its  temperature  is  raised  and  death  takes  place  in  from  48-72 
hours. 

Chronic  swine  erysipelas  is  the  less  severe  form  of  the  disease ;  recovery  after  an 
attack  is  not  infrequent,  though  some  animals  never  recover  completely.  When 
an  animal  begins  to  recover,  desquamation  occurs  about  the  spots  on  the  skin.  The 
characteristic  swelling  of  the  joints  is  responsible  for  a  peculiar  gait  noticed  in  infected 
animals,  and  for  the  disease  being  sometimes  called  Gout. 

Post  mortem,  in  swine  dead  of  the  disease,  there  is  frequently  in  addition  to  the 
spots  on  the  skin  an  intense  congestion  of  the  serous  membranes  and  of  the  intestines  ; 
the  lymphatic  glands  especially  those  of  the  abdomen  are  swollen  and  congested  ; 
the  spleen  is  very  much  enlarged  and  diffluent  and  shows  bosses  on  the  surface  ; 

1  [(Fr.  Rouget  du  pore,  erysipele,  rougeole.     Ger.  Schwein  Rothlauf.)J 


284  THE   BACILLUS   OF  SWINE   ERYSIPELAS 

the  liver  is  congested  and  the  blood  very  dark  in  colour.  More  rarely,  a  thickening 
of  the  walls  of  the  intestine  and  patches  of  broncho-pneumonia  are  present. 

The  specific  organism  is  found  in  the  liquid  discharges  from  the  bowel,  in  the 
spleen,  lymphatic  glands,  bone  marrow,  and  also  but  in  smaller  numbers  in  the 
blood,  liver  and  kidneys. 

The  bacillus  of  swine  erysipelas  appears  to  be  frequently  present  as  a 
saprophyte  in  the  tonsils  and  intestinal  canal  of  healthy  pigs  (Olt,  Pitt,  Over- 
beck).  Pitt  found  the  organism  in  the  intestine  in  26  out  of  66  and  in  the 
tonsil  in  28  out  of  50  normal  animals  examined  by  him. 

SECTION   I.— EXPERIMENTAL   INOCULATION. 
1.  Animals  susceptible  to  the  disease. 

Swine,  pigeons,  mice  and  rabbits  are  all  susceptible  to  swine  erysipelas 
but  in  different  degrees.  Guinea-pigs  are  immune. 

Inoculation  into  the  pectoral  muscle  of  a  pigeon  or  into  the  sub-cutaneous 
tissue  of  a  mouse  is  fatal  in  3  or  4  days.  Babbits  are  more  resistant  and  to 
produce  a  fatal  result  the  virus  must  be  inoculated  into  a  vein. 

When  passed  through  a  series  of  rabbits  the  virulence  of  the  virus  is  increased 
for  rabbits  but  diminished  for  swine.  The  first  rabbit  is  inoculated  intra-venously 
with  a  culture  from  a  pig  and  the  spleen  of  the  first  rabbit  is  used  for  inoculation 
of  the  second  and  so  on. 

On  the  other  hand,  the  virulence  is  increased  for  all  susceptible  animals  by 
passage  through  pigeons. 

It  is  a  curious  fact  that  swine  are  not  very  susceptible  to  experimental 
infection,  and  even  when  the  virus  is  inoculated  into  a  vein  it  seldom  leads 
to  a  fatal  result ;  so  that  to  produce  the  disease  in  these  animals  a  virus 
experimentally  increased  in  virulence  must  be  used  for  inoculation.  It  is 
however  possible  to  infect  pure  bred  swine  by  feeding  them  on  the  organs 
of  animals  which  have  died  of  the  disease. 

2.  Technique  of  inoculations. 

The  general  rules  applicable  to  the  inoculation  of  animals  must  be  observed 
and  special  attention  given  to  the  following  points.  The  material  for  inocula- 
tion may  be  taken  directly  from  the  spleen,  lymphatic  glands  or  blood  of  an 
animal  dead  of  the  disease,  though  it  is  better  to  sow  a  broth  culture  with  a 
fragment  of  the  spleen  and  to  inoculate  a  little  of  the  culture  after  incubating 
it  for  36-48  hours. 

3.  Symptoms  and  lesions. 

The  symptoms  have  already  been  detailed. 

The  most  prominent  lesion  in  experimentally-infected  animals  is  the 
swelling  and  softening  of  the  spleen.  The  organism  will  be  most  easily 
detected  in  the  spleen,  bone  marrow,  tonsils,  lymphatic  glands  and  blood, 
but  may  also  be  found  in  the  liver  and  kidneys.  Sections  of  the  lymphatic 
glands,  spleen,  liver  and  kidneys  should  also  be  cut. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  micro-organism  of  swine  erysipelas  is  a  small,  non-motile  bacillus, 
visible  only  with  difficulty  in  unstained  preparations,  and  measuring  0'5-1'5/* 
by  0'2-0'3/ji.  In  the  blood  and  internal  organs  it  occurs  singly,  in  pairs  or 


MORPHOLOGY  285 


in  groups.  In  broth  cultures  it  forms  short  chains  (fig.  182).  The  bacilli 
are  more  numerous  in  the  spleen  and  lymphatic  glands  than  in  the  blood. 
They  are  frequently  found  within  the  leucocytes,  and  in  sections  masses 
of  bacilli  will  be  seen  within  the  capillaries. 

The  bacillus  is  not  known  to  form  spores. 

Staining  methods.— The  bacillus  of  swine  erysipelas  stains  readily  with 


FIG.  182. — Bacillus  of  swine  erysipelas  (broth  FIG.    183. — Bacillus   of  swine  erysipelas  in 

culture).     Carbol-thionin.     (Oc.  iv,  obj.  T^th,  pigeon's   blood — Gram's  stain.     (Oc.  iii,  obi. 

Reichert.)  Ath,  Reichert.) 

the  basic  aniline  dyes,  is  gram-positive,  and  retains  the  violet  in  Claudius' 
method.  The  best  methods  to  use  are  : 

(a)  Cultures. — Stain  with  carbol-thionin  or  dilute  carbol-fuchsin. 

(6)  Blood-films  and  smears  of  tissues. — Carbol-thionin  or  carbol-methylene- 
blue  may  be  used,  but  Gram's  method  is  preferable. 

(c)  Sections. — Gram's  method  should  be  used  with  either  double  or  triple 
staining  (p.  219). 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  bacillus  of  swine  erysipelas 
is  indifferently  aerobic.  Growth  is  better  under  anaerobic 
conditions  but  is  always  rather  scanty. 

Cultures,  which  should  be  sown  with  the  blood,  pulp  of 
organs,  or  bone  marrow  of  an  animal  recently  dead  of  the 
disease,  are  easily  obtained  at  temperatures  between  15° 
and  40°  C.  on  the  ordinary  media. 

Broth.— The  medium  soon  becomes  slightly  opalescent 
when  incubated  at  33°-38°  C.  The  growth  which  is  always 
scanty  ceases  about  the  fourth  day,  and  subsequently  forms 
a  very  small  white  precipitate. 

Gelatin.  Stab  culture.— The  growth  in  gelatin  stab-culture 
is  characteristic.  Along  the  stab  a  thin  opaque  line  de- 
velops, from  which  numerous  small  very  delicate  branching 
filaments  radiate.  The  growth  is  more  luxuriant  in  the  depth 
of  the  stab.  Towards  the  twentieth  day,  the  characteristic 
appearance  vanishes  and  the  culture  becomes  cloudy.  There 
is  never  any  liquefaction  of  the  gelatin  (fig.  184). 

Stroke  culture. — Cultures  on  the  surface  of  a  sloped  Of  swine  erysipelas, 
gelatin  tube  radiate  from  the  line  of  sowing  like  the  feathers  gfJg'JjUS? in  gela" 
of  a  quill. 

Single  colonies.— Fine  downy  flocculi  giving  off  delicate  radiating  fila- 
ments are  seen  embedded  in  the  gelatin,  then  the  appearance  becomes 


286  THE   BACILLUS   OF   SWINE   ERYSIPELAS 

woolly-looking    and    the    centre    of    the    colony    forms    a    small    brownish 
spot. 

Agar. — At  first  the  growth  is  similar  to  that  on  gelatin,  but  it  soon  assumes 
an  homogeneous  appearance  and  forms  a  delicate 
scanty  layer. 

Potato. — On  potato  the  organism  only  grows  under 
anaerobic  conditions  and  then  forms  a  barely  visible 
streak. 

SECTION  III.— BIOLOGICAL   PROPERTIES. 
1.  Vitality  and  virulence. 

The  bacillus  of  swine  erysipelas  remains  alive  for 

FIG.  185.— Bacillus  of  swine    several  months  in  cultures  under  anaerobic  conditions, 
SatufSo  (I  dlys)COl°ny  °n    an(l  shows  an  equal  vitality  in  deep  stab  cultures  in 
ordinary   gelatin :    it   will   give  rise  to  sub-cultures 
and  even  kill  pigeons  after  6  months. 

In  aerobic  broth  cultures  kept  in  the  warm  (37°-39°  C.)  incubator  the 
virulence  as  well  as  the  vitality  vanish  much  more  rapidly.  The  virulence 
becomes  progressively  enfeebled  and  after  about  20  days  the  culture  is  harm- 
less. As  already  pointed  out  the  virulence  of  an  attenuated  virus  can  be 
restored  by  passage  through  pigeons. 

2.  Vaccination. 

One  attack  of  the  acute  disease  confers  immunity  on  swine  ;  moreover 
an  attack  of  the  chronic  form  (Gout)  protects  an  animal  from  the  acute  disease. 
Pasteur  and  Thuillier  considered  it  possible  to  immunize  animals  by  inoculating 
them  with  attenuated  viruses,  and  at  the  present  time  vaccination  of  swine 
is  very  extensively  practised,  especially  in  Austria. 

For  the  purposes  of  vaccination  broth  cultures  which  have  been  attenuated 
(through  the  action  of  the  oxygen  of  the  air)  by  incubation  for  a  longer  or 
shorter  time  in  the  warm  incubator  may  be  utilized. 

The  pigs  are  inoculated  first  with  a  very  weak  virus,  and  then  with  a  virus 
which  has  not  been  in  the  incubator  so  long  and  which  is  therefore  somewhat 
more  virulent.  Pigs  should  be  inoculated  before  they  are  4  months  old  as 
they  are  then  less  susceptible  to  the  disease.  The  immunity  so  conferred, 
which  is  complete  12  days  after  the  second  inoculation,  lasts  about  a  year, 
and  this  is  a  sufficient  length  of  time  for  fattening  purposes.  If  the  animal 
is  to  be  kept  for  breeding  it  is  well  to  repeat  the  vaccination  at  the  end  of  a 
year. 

As  stated  above,  the  organism  may  also  be  attenuated  for  the  pig  by  passage 
through  rabbits.  After  several  passages  the  virus  becomes  very  virulent  for  rabbits, 
but  attenuated  for  swine,  and  may  then  be  used  for  inoculation  as  a  vaccine.  For 
this  purpose  a  tube  of  broth  is  sown  with  the  spleen  of  the  last  rabbit  of  the  series 
and  incubated,  and  the  pig  is  vaccinated  with  the  culture. 

3.  Soluble  products.    Serum  therapy. 

(i)  Negative  results  follow  the  injection  of  filtered  cultures.  The  amount 
of  growth,  as  has  already  been  pointed  out,  always  remains  very  scanty  and 
toxins  are  not  formed  in  any  appreciable  quantity.  But  if  rabbits  be  inocu- 
lated sub-cutaneously  with  small  quantities  of  unfiltered  cultures  they 
quickly  recover,  and  it  soon  becomes  possible  to  inoculate  large  doses  into  the 
veins  without  producing  any  morbid  symptoms.  Emmerich,  Leclainche 
and  others  killed  rabbits  which  had  been  treated  in  this  manner,  made  an 


SERUM   THERAPY  287 

emulsion  by  pounding  and  extracting  the  muscles  and  organs,  and  obtained 
a  product  which  after  filtration  possessed  both  vaccinating  and  therapeutic 
properties. 

(ii)  Lorenz  prepares  a  serum  which  has  distinct  therapeutic  properties. 
A  rabbit  is  inoculated  sub-cutaneously  first  of  all  with  a  few  cubic  centi- 
metres of  specific  serum  (1  c.c.  per  1  kg.  of  body  weight)  ;  two  days  later, 
and  again  on  the  twelfth  day,  the  rabbit  is  inoculated  (sub-cutaneously)  with 
a  virulent  culture,  the  second  and  third  doses  being  larger  than  the  first. 
After  a  further  interval  of  10  days  a  large  dose  of  culture  is  administered 
intra-venously. 

(iii)  Mesnil,  adopting  Pasteur's  method  of  attenuated  viruses,  immunizes 
rabbits  as  follows  :  At  intervals  of  1  week  a  rabbit  receives,  first,  O25  c.c., 
then  1  c.c.  of  a  very  attenuated  virus — the  first  vaccine.  This  is  followed 
by  inoculations  of  a  less  attenuated  virus — the  second  vaccine — in  doses  of 
O25  c.c.  first  and  1  c.c.  later.  Finally,  at  periods  varying  from  a  week  to  a 
month  increasing  doses  of  virulent  culture — O25  c.c.,  1  c.c.,  3  c.c.,  4  c.c., 
5  c.c.,  10  c.c. — are  used.  Immunization  requires  about  6  months'  treatment, 
and  in  spite  of  the  small  doses  used  some  of  the  animals  die.  It  is  only  after 
about  3  months  that  the  animals  under  experiment  can  resist  the  inoculation 
of  a  large  dose  of  culture  every  week  or  10  days  without  showing  considerable 
reaction.  The  serum  of  animals  prepared  in  this  way  if  given  in  doses  of 
0'05  c.c.  protects  mice  against  an  inoculation  of  a  virulent  virus  given  the 
following  day.  In  doses  of  0'25  c.c.  it  exhibits  therapeutic  properties  provided 
it  be  administered  within  24  hours  of  infection.  The  serum  is  also  efficient 
in  the  case  of  pigeons  and  rabbits.  It  is  not  bactericidal,  for  if  the  bacillus 
be  sown  in  the  serum  it  grows  in  long  chains  and  moreover  retains  its 
virulence. 

(iv)  Leclainche  uses  horses  for  the  preparation  of  his  prophylactic  serum  : 
200  c.c.  of  a  virulent  culture  (one  which  will  kill  pigeons  in  doses  of  0'25  c.c. 
when  inoculated  into  the  pectoral  muscle)  are  inoculated  into  the  jugular 
vein  and  the  inoculations  repeated  at  intervals  of  about  10  days.  The 
resulting  serum  is  prophylactic  for  rabbits  in  doses  of  0*5-1  c.c.  and  the 
immunity  conferred  lasts  1  or  2  days.  Inoculation  of  1  c.c.  of  this  serum 
mixed  with  an  equal  volume  of  a  virulent  culture  confers  a  lasting  immunity 
(Sera-vaccination) . 

Leclainche  has  applied  this  method  to  the  vaccination  of  swine.  He  inoculates 
healthy  pigs  on  the  inner  side  of  the  thigh  first  with  a  mixture  of  5-10  c.c.  of  serum 
and  one-half  a  virulent  culture,  and  12  days  later  with  one-half  a  virulent  culture 
without  serum.  If  he  has  to  deal  with  an  herd  already  infected  he  inoculates  them 
with  10-20  c.c.  of  serum  without  any  culture  2  days  before  he  inoculates  the  first 
vaccine. 

The  serum  has  no  appreciable  therapeutic  property. 

4.   Agglutination. 

The  serums  prepared  by  Mesnil's  and  by  Leclainche's  methods  agglutinate 
the  bacillus  of  swine  erysipelas  to  a  marked  degree.  The  serums  of  infected 
rabbits  and  pigeons  show  no  agglutinating  properties  (Overbeck). 


SECTION  IV.— THE  DETECTION,   ISOLATION  AND   IDENTIFICATION 
OF  THE   ORGANISM. 

The  recognition  of  the  disease  is  of  supreme  importance  from  the  point  of 
view  of  vaccination,  and  the  demonstration  of  the   organism  is  of  great 


288  THE  BACILLUS   OF  SWINE  ERYSIPELAS 

help  in  diagnosis  and  in  differentiating  the  disease  from  hog  cholera  [swine 
fever]. 

The  following  observations  and  experiments  should  be  made  : 

1.  Stain  blood  films  and  smear  preparations  of  the  lymphatic  glands, 
spleen  and  bone  marrow  by  Gram's  method,  etc.  and  examine  them  for  the 
bacillus  (vide  "  Microscopical  appearance  "). 

2.  Sow  cultures  from  the  spleen  in  broth  and  on  gelatin. 

3.  Inoculate  pigeons  and  guinea-pigs  with  broth  cultures. 
Guinea-pigs  it  will  be  remembered  are  immune  to  swine  erysipelas,  but 

they  are  very  susceptible  to  hog  cholera  [or  swine  fever].     This  experiment 
therefore  will  differentiate  between  the  two  diseases. 

The  bacillus  of  mouse  septicaemia  (Koch). 

(Bacterium  murisepticum.) 

The  septicsemic  disease  of  the  domestic  mouse  (Mus  musculus),  investigated  by  Koch, 
is  due  to  a  small  bacillus,  similar  to  that  of  swine  erysipelas,  but  on  inoculation  somewhat 
less  rapidly  fatal  than  the  latter  organism.  It  is  not  pathogenic  for  the  field  mouse 
(Arvicola  arvalis)  nor  for  pigeons  nor  rabbits.  But  if  its  virulence  be  increased  by  numerous 
passages  through  mice  a  virus  is  ultimately  obtained  which,  on  intra-venous  inoculation, 
proves  fatal  to  pigeons. 

The  symptoms  observed  in  experimentally-infected  mice  are  drowsiness,  blepharitis 
and  a  spasmodic  form  of  respiration.  The  disease  finally  terminates  fatally.  Bacilli 
are  to  be  found  in  large  numbers  in  the  blood  and  in  the  internal  organs. 

The  morphological  characteristics  and  staining  reactions  of  this  organism  are  the  same 
as  those  of  the  bacillus  of  swine  erysipelas.  The  cultures  of  the  two  organisms  are  very 
much  alike ;  there  is,  however,  this  difference  that  the  growth  of  the  bacterium  murisep- 
ticum in  gelatin  is  more  cloudy  and  the  radiating  filaments  are  not  so  well  marked. 

The  serums  of  animals  immunized  against  the  bacillus  of  swine  erysipelas  agglutinate 
the  bacillus  of  mouse  septicaemia  (Overbeck).  This  affords  definite  proof  that  the  latter 
bacillus  is  not  a  distinct  species  [but  merely  a  variety  of  the  bacillus  of  swine  erysipelas 
adapted  to  its  special  host.] 


CHAPTER  XVIII. 
BACILLUS  TUBERCULOSIS. 

Introduction. 

1.  Types  of  tubercle  bacilli,  p.  289.     2.  Human  tuberculosis,  p.  292.     3.  Tuber- 
culosis in  the  lower  animals,  p.  294.     4.  Associated  micro-organisms,  p.  297. 
Section  I. — Experimental  inoculation,  p.  297. 
Section  II. — Morphology,  p.  305. 

1.  Microscopical  appearance,  p.  305.     Staining  methods,  p.  306.     The  staining  of 
films,  p.  307.     The  staining  of  sections,  p.  310.     Appearance  of  stained  bacilli,  p.  312. 
2.  Cultural  characteristics,  p.  314. 
Section  III. — Biological  properties,  p.  322. 

1.  Viability  and  virulence,  p.  322.  The  action  of  antiseptics,  p.  323.  2.  Toxins, 
p.  323.  The  toxic  properties  of  dead  bacilli,  p.  323.  Koch's  old  tuberculin,  p.  324. 
Tuberculins  T.A.,  T.O.,  T.R.,  p.  328.  Maragliano's  tuberculin,  p.  329.  Toxalbumin, 
p.  329.  3.  Vaccination,  p.  330.  4.  Serum  therapy,  p.  334.  5.  Agglutination,  p.  335. 
6.  Immune  body,  p.  337. 
Section  IV. — The  detection  of  the  tubercle  bacillus,  p.  337. 

A.  Sputum,  p.  339.  B.  Blood,  p.  341.  C.  Pus,  p.  342.  D.  Exudates,  p.  342. 
E.  Granulomata,  p.  343.  F.  Nasal  cavities,  p.  343.  G.  Urine,  p.  343.  H.  Ex- 
creta, p.  344.  I.  Milk,  p.  345. 

The  paratubercle  or  acid-fast  bacilli,  p.  345. 

The  smegma  bacillus,  p.  346.  The  bacillus  of  verruga  peruana,  p.  346.  Pseudo- 
tuberculoses,  p.  347. 

THE  tubercle  bacillus,  discovered  by  Koch,  is  the  cause  of  tuberculosis  in 
man  and  the  lower  animals. 

In  accordance  with  established  practice,  the  infecting  agent  in  tuberculosis  will, 
in  the  present  chapter  and  elsewhere  in  this  book,  be  spoken  of  as  a  bacillus  although 
it  is  now  agreed  that  it  should  be  classed  with  the  genus  Discomyces  (Streptothrix 
of  Cohn).  Metchnikoff  has  suggested  for  it  the  name  Sclerothrix  kochi. 

1.  Types  of  tubercle  bacilli. 

Of  the  tubercle  bacilli  recoverable  from  human  tissues  and  from  the  tissues 
of  the  lower  animals  four  types  can  be  distinguished,  differing  from  one 
another  in  various  characteristics.  It  is  customary  therefore  to  speak  of 
human,  bovine,  avian  and  ichthic  tubercle  bacilli,  meaning  thereby  the 
type  of  bacillus  [most  commonly]  obtained  from  human,  bovine,  avian  or 
ichthic  sources  respectively. 

(a)  The  human  and  the  bovine  types  of  tubercle  bacilli. — Most  bacterio- 
logists, Koch's  opinion  notwithstanding,  are  agreed  in  regarding  the  human 
and  the  bovine  types  of  tubercle  bacilli  as  identical,  for  [in  the  opinion  of  these 
observers  ]  the  facts  which  can  be  brought  in  support  of  this  view  are  numerous 
and  conclusive.  Each  bacillus  though  best  adapted  to  the  particular  species 


290  THE   TUBERCLE   BACILLUS 

of  animal — man  and  bovine  respectively — in  which  it  finds  its  normal  habitat 
may  nevertheless  infect  either  of  them,  and  though  the  bovine  bacillus  appears 
to  be  more  virulent  than  the  human  bacillus  the  latter,  according  to  de  Jong, 
may  by  passage  through  goats  be  made  as  virulent  for  bovine  animals  as 
the  bovine  bacillus  itself. 

[The  findings  of  the  English  Commission l  are  not  altogether  in  agreement 
with  the  statements  contained  in  the  preceding  paragraph. 

[In  the  opinion  of  this  Commission  the  human  and  bovine  types  are  not 
identical  but  "varieties  of  the  same  bacillus."  They  point  out  that  since 
the  human  and  the  bovine  tubercle  bacilli  are  "  morphologically  indistin- 
guishable "  the  question  of  their  identity  or  non-identity  resolves  itself  into 
a  consideration  of  their  cultural  and  pathogenic  differences  or  similarities. 

[With  regard  to  the  former,  the  human  type  consistently  grows  more 
luxuriantly  in  culture  than  the  bovine  type  and  this  difference  in  cultural 
characteristics  is  quite  definite  though  "  the  gap  which  separates  the  human 
type  from  those  strains  of  the  bovine  type  which  grow  most  abundantly  is 
not  wide." 

[A  study  of  their  pathogenic  resemblances  and  differences  shows  on  the 
one  hand  that  the  disease  produced  in  certain  species  of  animals  such  as 
guinea-pigs  and  monkeys  by  the  two  types  is  "  histologically  and  anatomi- 
cally identical  "  and  on  the  other  hand  that  in  man  fatal  tuberculosis  due 
to  infection  with  bacilli  of  the  bovine  type  is  identical  with  that  caused  by 
the  human  type. 

[That  the  bovine  bacillus  can  infect  man  is  certain.  Many  cases  of  tubercu- 
losis in  children  and  a  few  in  adults  investigated  by  Cobbett  and  A.  S.  Griffith 
(working  for  the  English  Commission)  were  shown  to  be  caused  solely  by  the 
bovine  tubercle  bacillus.  An  infection  of  the  bovine  species  by  the  human 
tubercle  bacillus  on  the  other  hand  did  not  occur  :  the  human  tubercle 
bacillus  was  in  fact  incapable  of  producing  in  cattle  anything  but  a  slight 
and  non-progressive  tuberculosis,  however  large  the  dose. 

[Neither  did  the  human  type  of  bacillus  cause  anything  more  than  a  slight 
non-progressive  tuberculosis  in  goats,  pigs  and,  with  rare  exceptions,  in  rabbits, 
while  the  bovine  bacillus  readily  caused  a  fatal  tuberculosis  in  these  animals 
as  well  as  in  cattle. 

[Certain  tubercle  bacilli  isolated  during  the  investigations  of  the  Com- 
mission from  cases  of  lupus  and  equine  tuberculosis  had  the  cultural  charac- 
teristics of  the  bovine  bacillus  but  were  only  slightly  virulent  for  calves  and 
rabbits  (the  animals  usually  relied  on  for  differential  tests)  and  were  of 
relatively  low  virulence  also  for  monkeys  and  guinea-pigs.  These  bacilli, 
it  would  seem,  in  no  way  bridge  the  gap  between  the  two  types  ;  for  while 
they  approach  the  human  tubercle  bacillus  in  their  low  degree  of  virulence 
for  calves  and  rabbits,  they  recede  from  it  in  virulence  for  monkeys  and 
guinea-pigs  (A.  S.  Griffith).  At  the  same  time,  as  the  Commissioners  point 
out,  "  the  discovery  of  these  exceptional  bacilli  makes  it  impossible  to  regard 
differences  of  virulence  for  the  calf  and  rabbit  as  sufficient  to  establish  the 
non-identity  of  the  human  and  bovine  types."  Several  of  these  attenuated 
bacilli  isolated  from  human  (lupus)  and  equine  sources  were  raised  to  the 
full  virulence  of  a  typigal  bovine  bacillus  by  passage  through  calves  and 
rabbits. 

[To  establish  the  complete  identity  of  the  two  types  it  would  appear  to  be 
necessary  to  demonstrate  that  both  cultural  and  pathogenic  differences  were 
unstable,  i.e.  that  the  transmutation  of  the  human  type  of  bacillus  into  the 

[x  The  references  to  the  "  English  Commission  "  in  this  chapter  are  to  the  Reports 
and  Appendices  thereto  of  the  Royal  Commission  on  Tuberculosis  appointed  in  1901.] 


bov 


TYPES   OF   TUBERCLE   BACILLI  291 


bovine  or  vice  versa  was  possible,  and  on  this  point  after  reviewing  the  numerous 
prolonged  passage  experiments  on  various  species  of  animals  carried  out  under 
their  direction  the  Commissioners  conclude  that  "  transmutation  of  bacillary 
type  "  is  "  exceedingly  difficult  if  not  impracticable  of  accomplishment  by 
laboratory  procedure." 

[Though  it  has  been  considered  desirable  to  introduce  thus  briefly  the  conclusions 
arrived  at  by  the  English  Commission,]  it  is  altogether  beyond  the  scope  of  the 
present  work  to  enter  upon  a  discussion  of  the  arguments  which  have  been  brought 
forward  in  support  of  their  theses  by  those  who  hold  that  the  human  tubercle  bacillus 
is  identical  with  the  bovine  and  by  those  who  are  of  contrary  opinion.  For  these 
arguments  the  reader  is  referred  to  the  publications  of  the  authors  whose  names 
are  mentioned  in  the  text  and  to  those  of  other  writers  on  the  subject. 

(6)  The  avian  tubercle  bacillus.— Straus  and  Gamaleia  regard  avian  tuber- 
culosis as  due  to  a  special  organism  which,  though  closely  allied  to  the  human 
bacillus,  constitutes  a  separate  species.  The  view  long  held  by  Arloing  and 
others  that  the  human  and  the  avian  bacillus  are  identical  has  been  [held  to 
be]  proved  by  certain  experiments  of  Nocard  (vide  infra).  [It  is  largely  on 
the  results  of  these  latter  experiments  that]  the  bacillus  of  avian  tuberculosis 
has  been  regarded  merely  as  a  strain  or  race  of  the  human  tubercle  bacillus. 

Nocard  [claims  to  have]  converted  an  human  tubercle  bacillus  into  an  avian  tubercle 
bacillus  by  growing  it  for  a  long  time  in  collodion  sacs  in  the  peritoneal  cavities  of 
fowls.  Nocard  filled  a  collodion  sac  (p.  175)  with  a  thick  emulsion  of  a  glycerin- 
potato  culture  of  a  human  bacillus.  The  sac,  after  remaining  at  least  4  months  in 
the  peritoneal  cavity  of  a  fowl,  contained  a  thick  mass  of  bacilli  which,  when  sown 
on  culture  media,  gave,  at  first,  a  scanty  growth,  and  this  on  sub-culture  became 
more  luxuriant  and  had  all  the  characteristics  of  a  culture  of  the  avian  bacillus  (a 
soft,  greasy,  fatty,  easily  dissociated  and  wrinkled  layer  of  growth).  These  cultures 
were  only  slightly  virulent  for  guinea-pigs  but  highly  virulent  for  rabbits  which 
succumbed  to  a  generalized  miliary  tuberculosis  on  inoculation  with  bacilli  from 
the  first  passage  and  when  inoculated  with  bacilli  from  the  second  passage  the 
animal  died  of  a  tuberculous  septicaemia  without  apparent  lesions  exactly  as  though 
it  had  been  inoculated  with  an  avian  bacillus.  After  three  passages  of  6-8  months 
in  collodion  sacs  the  human  tubercle  bacillus  killed  fowls  with  symptoms  identical 
with  those  of  the  spontaneous  disease. 

[A.  S.  and  F.  Griffith  (working  for  the  English  Commission)  entirely  failed  to 
confirm  the  results  obtained  by  Nocard.  No  modification  of  human  or  bovine 
tubercle  bacilli  into  avian,  or  of  avian  tubercle  bacilli  into  mammalian,  was 
demonstrated. 

["  With  ten  mammalian  viruses,  eight  of  which  were  bovine,  sixteen  collodion 
capsule  experiments  on  fowls  and  twenty  on  pigeons  were  performed,  lasting  55-186 
days.  In  certain  of  the  cases  cultures,  which  were  obtained  from  the  capsules  on 
removal  from  the  bird's  peritoneal  cavity,  were  placed,  again  in  capsules,  in  the 
peritoneal  cavities  of  other  birds,  the  total  duration  of  residence  being  in  one  series 
as  much  as  475  days.  In  20  of  these  experiments  cultures  were  obtained  from  the 
capsules  and  found  to  be  unchanged  in  character.  In  the  remaining  16  cases  the 
bacilli  in  the  capsules  were  apparently  dead." 

["  Similar  experiments  were  performed  with  human  tubercle  bacilli  obtained 
from  12  different  sources.  These  experiments  lasted  from  59  to  685  days."  The 
results  were  similar  to  those  obtained  with  mammalian  tubercle  bacilli. 

["  With  cultures  of  five  avian  viruses  25  collodion  capsule  experiments  were 
performed  on  guinea-pigs.  The  duration  of  residence  in  individual  guinea-pigs 
ranged  up  to  253  days  and  the  total  periods  during  which  the  cultures  were  in  the 
peritoneal  cavities  of  series  of  guinea-pigs  varied  up  to  424  days."  In  two  instances 
the  bacilli  in  the  capsules  were  dead  :  "  from  all  the  other  capsules  cultures  were 
obtained  and  the  bacilli  were  found  to  be  unchanged  "  in  cultural  characteristics 
and  virulence.  ] 

Lydia  Rabinowitsch  isolated  thirty-four  strains  of  tubercle  bacilli  from  birds. 
Two  of  these,  isolated  from  birds  of  prey,  had  all  the  characteristics  of  the  human 
bacillus.  Rabinowitsch  concluded  from  this  investigation  that  the  human  and 


292  THE   TUBERCLE   BACILLUS 

avian  tubercle  bacilli  are  merely  two  varieties  of  the  same  species  adapted  to  different 
conditions. 

(c)  The  ichthic  tubercle  bacillus. — The  ichthic  tubercle  bacillus  is  more 
sharply  differentiated  from  its  congeners  (vide  infra)  but  some  observers, 
notably  Moeller,  Sorgo  and  Suess  [report  that  they]  have  succeeded  in  con- 
verting an  human  into  an  ichthic  tubercle  bacillus. 

It  appears  to  be  true  that  all  tubercle  bacilli  have  a  common  origin,  and  that 
acclimatization  under  parasitic  conditions  in  different  animals  has  led  to  the 
creation  of  the  four  different  types. 

2.  Human  tuberculosis. 

Man  becomes  infected  with  tuberculosis  either  by  way  of  the  respiratoi 
or  digestive  tracts,  more  rarely  by  the  skin  and  genital  passages. 

The  tubercle  bacillus  is  found  in  all  tuberculous  lesions  in  the  hums 
subject. 

[As  to  the  aetiology  of  human  tuberculous  phthisis  opinion  is  somewhat 
sharply  divided.  The  original  theory  was  that  tuberculous  phthisis  is  com- 
monly caused  by  the  inhalation  of  tubercle  bacilli.  This  doctrine  received 
the  support  of  Koch,  Cornet,  Pfliigge  and  others.  Chauveau  however  put 
forward  the  view  many  years  ago  that  phthisis  was  not  uncommonly  caused 
by  bacilli  which  had  been  ingested  and  absorbed  from  the  intestine,  and  in 
recent  years  this  doctrine  has  been  strongly  advocated  by  Behring.] 

Behring  thinks  that  infection  generally  takes  place  through  the  alimentary 
canal  and  that  pulmonary  tuberculosis  of  adults  is  merely  a  later  stage  of  an 
intestinal  infection  contracted  in  the  early  years  of  life.  Calmette  and 
Guerin  confirm  this  opinion  [in  so  far  as  it  relates  to  the  channel  of  infection 
in  phthisis ]  ]  and  bring  forward  numerous  experiments  to  show  that  pulmonary 
tuberculosis  (not  inoculated)  is  always  a  sequela  of  a  primary  intestinal 
infection  of  which  in  the  adult  no  trace  of  the  original  lesions  in  the  mesenteric 
glands  or  abdominal  viscera  can  be  detected.  [Calmette  bases  his  opinion 
upon  experiments  on  goats  and  bovines  and  on  the  researches  of  his  pupils, 
Van  Steenberghe  and  Grysez,  on  experimental  anthracosis.  E-avenel  also 
from  his  own  observations  is  led  to  believe  that  infection  of  the  tonsils  is  the 
most  frequent  cause  of  apical  tuberculosis  but  that  infection  may  take  place 
from  any  part  of  the  alimentary  canal  and  that  the  bacilli  may  pass  through 
the  wall  of  the  intestine  without  leaving  any  indication  of  the  site  of  infection 
in  the  form  of  a  local  lesion. 

[The  view  that  phthisis  is  commonly  caused  by  the  inhalation  of  tubercle 
bacilli  is,  however,  supported  by  many  recent  investigations.  Cobbett,  for 
instance,  believes  "  that  phthisis  is  commonly  caused  by  the  inhalation  of 
tubercle  bacilli "  and  from  the  results  of  an  elaborate  series  of  experiments 
devised  to  ascertain  the  aetiology  of  pulmonary  tuberculosis  in  which  he 
repeats  many  of  Calmette's  experiments  this  observer  concludes  that  the 
intestine  is  not  a  common  portal  of  entry  for  the  tubercle  bacilli  which  cause 
phthisis.  The  experiments  of  the  English  Commission  again  though  they 
demonstrate  "  that  a  considerable  amount  of  the  tuberculosis  of  childhood 
is  to  be  ascribed  to  infection  with  bacilli  of  the  bovine  type  transmitted  to 
children  in  meals  consisting  largely  of  milk  of  the  cow  "  nevertheless  do  not 
entirely  support  the  theory  that  pulmonary  tuberculosis  is  a  sequela  of  a 
primary  intestinal  infection  as  may  be  seen  from  the  widely  different  propor- 

[l  Calmette  does  not  state  that  the  infection  in  pulmonary  tuberculosis  is  necessarily 
an  infantile  infection  but  merely  that  at  whatever  age  infection  of  the  lungs  occurs  the 
channel  of  infection  is  invariably  the  alimentary  canal.] 


HUMAN  TUBERCULOSIS 


293 


tion  of  bovine  and  human  tubercle  bacilli  found  respectively  in  alimentary 
and  in  pulmonary  lesions  in  man.  Thus  of  nine  cases  of  cervical  gland 
tuberculosis  in  children  three  were  found  to  be  caused  by  the  bovine  tubercle 
bacillus  and  six  by  the  human  tubercle  bacillus  ;  and  of  twenty-seven  cases 
of  primary  abdominal  tuberculosis  in  children,  fourteen  were  caused  by  the 
bovine  tubercle  bacillus  and  thirteen  by  the  human  tubercle  bacillus  (Cobbett 
and  A.  S.  Griffith).  "  In  these  cases,"  the  Commission  remarks,  "  the  tubercle 
bacillus  had  unquestionably  been  swallowed."  The  examination  of  tissues 
from  fourteen  fatal  cases  of  primary  pulmonary  tuberculosis  (A.  S.  Griffith 
and  Cobbett)  showed  that  in  all  of  the  cases  the  human  tubercle  bacillus 
alone  was  responsible  for  the  disease.  A.  S.  Griffith  subsequently  examined 
the  sputum  from  twenty-eight  cases  of  pulmonary  tuberculosis  :  in  twenty- 
six  the  human  tubercle  bacillus  was  the 
infective  agent  and  in  the  remaining  two 
the  bovine  tubercle  bacillus  (confirmed  by 
repeated  examination  of  the  sputum). 

[Baumgarten  holds  that  tuberculous 
phthisis  is  due  to  infection  during  intra- 
uterine  life,  but  this  view  receives  very 
little  support  at  the  present  day.] 

Attempts  have  been  made  to  draw  a  dis- 
tinction between  the  disease  as  it  affects  the 
internal  organs,  pleurae  and  peritoneum  on  the 
one  hand  and  that  form  of  it  which  affects 
the  skin,  glands,  joints,  etc.  According  to 
Arloing,  the  latter,  the  so-called  surgical  tuber- 
culoses, are  due  to  an  attenuated  bacillus  which 
must  be  regarded  as  a  separate  variety.  But 
seeing  that  in  these  localized  lesions  the  bacillus 
is  fully  virulent,  it  is  more  likely  that  the  slight 
tendency  to  dissemination  which  it  exhibits  is 
to  be  explained  on  other  grounds,  such  as  the 
personal  resistance  of  the  infected  individual, 
the  influence  of  the  particular  tissues  in  which 
it  is  growing,  and  the  small  number  of  the 
invading  organisms  which  grow  but  feebly  in 
a  soil  relatively  unfavourable  to  their  multipli- 
cation (Krompecher  and  Zimmermann). 

[ Arloing' s  theory,  in  so  far  as  it  relates  to 
tubercle  bacilli  which  infect  the  skin,  is  in  part 
supported,  and  greatly  amplified,  by  A.  S. 
Griffith  (working  for  the  English  Commission). 
Twenty  cases  of  lupus  were  examined.  The 
tubercle  bacilli  isolated  from  nine  of  them  showed 
the  cultural  characteristics  of  the  bovine 
tubercle  bacillus,  but  only  one  had  the  patho- 
genicity  ordinarily  associated  with  that  type, 
while  the  rest  showed  varying  degrees  of  lesser 

virulence:  the  least  virulent  being  no  more  dysgonic  or  bovine  type :  (ft)  the  eugonicor 
virulent  for  calves  and  rabbits  than  a  human  human  type.  (This  and  the  succeeding 
tubercle  bacillus  but  differing  from  the  latter  $"7$$  ^mSion6  o^TubSosis 
in  that  they  were  also  of  relatively  slight  (Human  and  Bovine) — Part  II.  Appendix, 
virulence  for  guinea-pigs  and  monkeys.  Ssio^oUheCon'tro&fH'KTa&n^ 

L-brom  the  remaining  eleven  cases  tubercle    office.) 
bacilli  were  isolated  which  had  the  cultural 

characteristics  of  the  human  tubercle  bacillus ;  two  had  the  full  virulence  of  the 
human  tubercle  bacillus,  the  others  being  of  lower  virulence. 

[It  was  found  possible  in  two  of  the  cases  in  which  a  degraded  bovine  bacillus 
was  the  infective  agent  to  "  increase  the  virulence  of  the  culture  from  the  original 


10th  Generation 
3  months  o!d. 


(b) 

5th  Generation 
56  days  old. 


294 


THE  TUBERCLE  BACILLUS 


(a) 

8th  Generation 
4  months  old. 


(W 

5th  Generation. 
28  days  old. 


FIG.  187. — Tubercle  bacilli  from  cases  of 
Lupus  growing  on  glycerin-potato,     (a)  The 
nic  or  bovine  type:   (6)  the   eugonic 


type.     (A.    S.    Griffith.)      (See 


material  by  residence  in  the  tissues  of  calf  and  rabbit  so  as  to  bring  it  up  to  the 
high  virulence  of  the  bovine  tubercle  bacillus  "  :  and  one  of  the  strains  of  degraded 
human  tubercle  bacilli  attained  the  full  virulence  of  the  human  tubercle  bacillus 

after  residence  in  the  body  of  a  monkey. 
No  correspondence  suggesting  any  rela- 
tion between  the  duration  or  extent  of 
the  disease  in  the  human  patient  and  the 
degree  of  attenuation  of  the  bacillus  iso- 
lated was  demonstrable  in  these  cases. 

[From  thirteen  cases  of  joint  and  bone 
tuberculosis  the  human  tubercle  bacillus 
with  the  full  virulence  of  the  type  alone 
was  isolated ;  in  a  fourteenth  case  both 
human  and  bovine  tubercle  bacilli  appear 
to  have  been  present  (Cobbett  and  A.  S. 
Griffith).  These  investigations  therefore 
afford  no  confirmation  of  Arloing's  theory 
so  far  as  it  applies  to  joint  or  gland 
tuberculosis.  ] 

3.  Tuberculosis  in  the  lower 
animals. 

The  majority  of  the  lower  animals  are 
susceptible  to  infection  with  tuberculosis ; 
[The  infecting  agent  however  is  not 
always  of  the  same  type.] 

Bovine  animals. — Adult  animals  are 
frequently  tuberculous  (3-60  per  cent, 
varying  according  to  the  locality), 
[young]  calves  very  rarely  so  (1  in 
10,000  at  the  most). 

Generally  speaking  the  disease  runs  a  chronic  course.  Cattle  may  suffer  from  the 
disease  for  a  long  time  without  showing  any  loss  of  weight. 

The  respiratory  organs  are  most  frequently  affected  :  large,  occasionally  calci- 
ned, tuberculous  masses  (Grapes  x)  are  found  in  the  lungs :  the  pleurae  and  especially 
the  bronchial  glands  are  affected  at  the  same  time :  occasionally  the  abdomen 
(mesenteric  glands,  liver  and  more  rarely  the  spleen  and  kidneys)  is  invaded.  Some- 
times, especially  in  young  cattle,  the  disease  is  mainly  confined  to  the  alimentary 
tract :  the  lymphoid  structures  of  the  intestine,  the  mesenteric  glands,  peritoneum, 
liver  and  spleen  being  infected.  Other  local  manifestations  of  the  disease  are  some- 
times found  in  cattle  ;  for  instance,  mammary  tuberculosis  (in  about  1  per  cent, 
of  tuberculous  animals),  tuberculosis  of  bone,  etc. 

Finally,  bovine  tuberculosis  may  occur  as  a  rapidly-spreading  generalized  infection 
resembling  the  miliary  tuberculosis  of  man. 

[The  bovine  type  of  tubercle  bacillus  has  been  shown  to  be  the  sole  cause  of 
bovine  tuberculosis.] 

Monkeys. — In  these  climates,  monkeys  frequently  develop  tuberculosis, 
and  in  them  the  disease  runs  a  course  similar  to  that  of  human  tuberculosis, 
a  characteristic  feature  being  its  tendency  to  become  generalized.  In  these 
animals  the  commonest  form  of  the  disease  is  tuberculosis  of  the  lung  [and 
appears  to  be  due  mainly  to  the  human  type  of  tubercle  bacillus  (Rabino- 
witsch).  Thus  of  twenty-seven  cases  of  tuberculosis  in  monkeys  the  human 
type  of  bacillus  was  found  in  nineteen  and  the  bovine  type  in  three  :  the 
avian  type,  or  modified  organisms  or  mixtures  of  different  types  were  found 
in  the  remaining  five]. 

Dogs. — Tuberculosis  is  not  uncommon  among  dogs  (Cadiot),  though  the 
fact  has  for  a  long  time  remained  unrecognized.  In  dogs  the  lesions  often 
t1  Fr.  Pommeliere  ;  Ger.  Perlsucht.] 


dysgo 

or   human 

fig.  186.) 


TUBERCULOSIS  IN  ANIMALS  295 

simulate  malignant  growths  and  they  have  been  mistaken  for  neoplasms. 
Sometimes  however  they  resemble  the  lesions  found  in  man  and  this  is  especi- 
ally true  in  cases  where  cavitation  of  the  lungs  has  been  produced. 

Pigs. — Of  pigs  killed  in  public  slaughter  houses  [in  France]  one  to  ten  per 
thousand  are  infected  with  tuberculosis. 

As  a  general  rule,  the  alimentary  tract  is  the  part  affected.  Tuberculous 
otitis  has  been  recorded  in  pigs  :  when  it  occurs  it  is  probably  secondary  to 
some  pharyngeal  lesion  which  has  spread  up  the  Eustachian  tube.  Tuber- 
culosis of  the  respiratory  passages  and  localized  tuberculous  foci  are  not 
often  seen.  The  disease  is  sometimes  of  a  miliary  type  and  runs  a  rapid 
course. 


v  - 


•u,»« 


FIG.  188.— Section  of  the  liver  of  a  pig  which  died  47  days  after  intra-venous 
inoculation  with  50  mg.  of  culture  of  avian  tubercle  bacilli.  This  area  is  typical 
of  the  condition  found  in  the  liver  of  this  animal.  Note — (1)  the  profuse 
growth  of  bacilli,  with  tendency  to  rosette  formation  ;  (2)  the  huge  "  giant 
cell "  showing  multiplication  of  nuclei  by  irregular  longitudinal  splitting ; 
(3)  the  absence  of  wandering  cells,  with  the  exception  of  a  few  small  lympho- 
cytes ;  (4)  the  presence  of  numerous  bacilli  in  the  blood  stream,  x  600. 
(Eastwood.)  i 

[The  nature  of  the  tubercle  bacilli  occurring  in  fifty-nine  cases  of  natural 
tuberculosis  in  swine  was  investigated  by  A.  S.  Griffith  and  F.  Griffith  (for 
the  English  Commission).  Of  these,  fifty  (including  thirty- three  cases  of 
generalized  tuberculosis)  were  shown  to  be  due  to  the  bovine  tubercle  bacillus  ; 
three  (in  which  the  disease  was  localized  in  the  sub -maxillary  glands)  were 
caused  by  the  human  tubercle  bacillus  ;  five  (in  which  the  disease  was  similarly 
localized)  by  the  avian  tubercle  bacillus  ;  and  from  one  (localized  tubercu- 
losis) a  mixed  culture  of  avian  and  bovine  tubercle  bacilli  was  obtained. 

[Severe  and  generalized  tuberculosis  in  the  pig  therefore  was  by  this 
investigation  shown  to  be  due  to  the  bovine  tubercle  bacillus  only.] 

Rabbits. — There  is  no  foundation  in  fact  for  the  popular  belief  that  rabbits 
are  very  commonly  tuberculous.  Spontaneous  tuberculosis  in  the  rabbit  is, 
on  the  contrary,  a  comparatively  rare  disease.  When  it  occurs  it  assumes 
the  pulmonary  form. 

Goats  and  sheep. — Both  goats  and  sheep  are  liable  to  infection  with  tuber- 
culosis but  the  disease  in  these  animals  is  uncommon. 

['This  figure  as  well  as  figures  191,  192,  193,  198,  199,  200,  201,  202,  203,  205  and  206 
are  from  the  Final  Report  of  the  Royal  Commission  on  Tuberculosis  (Human  and 
Bovine)— Part  II.  Appendix,  Vol.  V. ;  Dr.  Arthur  Eastwood — by  permission  of  the  Con- 
troller of  H.M.  Stationery  Office.]  • 


296  THE   TUBERCLE   BACILLUS 

Horses. — Tuberculosis  is  rarely  seen  in  horses.  When  it  occurs  it  is  generally 
of  the  abdominal  type.  A  pulmonary  infection  is  occasionally  seen  which 
may  assume  the  character  of  a  miliary  tuberculosis  or  of  diffuse  infiltration 
of  the  lung,  and  large  sarcoma-like  masses  may  also  occur. 

[F.  Griffith  (for  the  English  Commission)  investigated  five  cases  of  equine 
tuberculosis.  From  three  of  these  bovine  tubercle  bacilli  of  standard  viru- 
lence were  isolated ;  the  bacilli  obtained  from  the  remaining  two  had  the 
cultural  characteristics  of  the  bovine  tubercle  bacillus  associated  with  a  low 
degree  of  virulence  for  all  the  test  animals — calf,  rabbit,  monkey,  guinea- 
pig,  etc.  By  prolonged  passage  experiments  the  virulence  of  the  latter 
bacilli  was  increased  to  that  of  the  bovine  tubercle  bacillus.] 

Cats. — Cats  are  rarely  tuberculous  but  when  the  disease  occurs  the  lesions 
are  similar  to  those  seen  in  dogs.  The  commonest  form  is  a  localized  infection 
of  the  alimentary  canal.  [Investigations  by  A.  S.  Griffith  and  F.  Griffith 
show  that  the  bovine  tubercle  bacillus  is  the  cause  of  natural  tuberculosis 
in  the  cat.] 

Birds.— Tuberculosis  is  a  very  common  disease  among  birds  :  fowls, 
pheasants,  guinea-fowl,  partridges,  peacocks,  parrots,  birds  of  prey,  etc. 
are,  all  of  them,  very  frequently  infected.  [The  disease  sometimes  appears 
as  a  rapidly  fatal  epizootic  among  farm-yard  fowls.] 

Tuberculosis  in  birds  is  usually  primary  in  the  alimentary  tract  developed  [it  is 
affirmed]  as  a  result  of  swallowing  the  excreta  of  tuberculous  animals  or  infected 
human  sputum. 

[The  investigations  of  the  English  Commission  do  not  support  this  view  of  the 
setiology  of  avian  tuberculosis.  Their  experiments  would  tend  to  show  that  birds 
(excluding  the  parrot)  are  not  susceptible  to  mammalian  tubercle  bacilli.] 

Tuberculosis  in  parrots  is  often  associated  with  a  bacillus  of  the  human  type 
and  is  due  to  infection  from  the  human  subject  (Eberlein,  Cadiot,  Straus).  From 
the  experimental  point  of  view  parrots  are  most  easily  infected  with  the  human 
tubercle  bacillus,  next  with  the  bacillus  of  the  bovine  type,  they  appear  to  be 
least  susceptible  to  the  avian  type. 

In  birds  the  liver  and  spleen  are  the  organs  most  commonly  affected  :  pulmonary 
lesions  are  rare  though  the  lungs  may  become  infected  in  the  last  stages  of  the  dis- 
ease. Except  in  parrots,  tuberculosis  of  the  skin,  mucous  membranes  or  joints 
is  rarely  seen.  The  disease  may  be  congenital  in  origin  the  egg  becoming  infected 
in  the  oviduct  (Baumgarten,  L.  Rabinowitsch,  Weber  and  Bofinger). 

The  histological  appearances  of  tuberculous  lesions  in  birds  are  unlike  those  in 
mammals  :  and  moreover  present  different  features  in  the  various  species.  Not 
uncommonly  the  viscera  will  be  found  to  be  infiltrated  with  bacilli  while  there  are 
no  visible  tubercles. 

Gold-blooded  vertebrata. — Tuberculous  lesions  have  been  found  in  the  boa- 
constrictor,  the  python,  the  ringed  snake  [Coluber  natrix — Linn.  ]  and  the 
frog.  Dubard  investigated  a  tuberculous  condition  in  the  carp  caused  by  a 
bacillus  apparently  very  closely  related  to  the  human  bacillus. 

The  bacillus  of  ichihic  tuberculosis  is  very  similar  to  the  human  tubercle  bacillus 
except  that  it  grows  badly  at  temperatures  above  25°  C.  and  in  this  respect  resembles 
the  para-tubercle  or  acid-fast  bacilli  (vide  infra}. 

Cultures  obtained  from  the  carp  are  pathogenic  to  frogs,  toads,  lizards,  tortoises, 
adders,  the  common  grass  snakes,  carp  and  other  fish  of  the  same  genus,  etc.  The 
bacilli  are  non-pathogenic  to  guinea-pigs  and  birds  ;  but  by  passage  through  guinea- 
pigs  the  organism  becomes  virulent  for  that  rodent.  Ichthic  tubercle  bacilli  when 
inoculated  into  rabbits  or  guinea-pigs  behave  in  the  same  way  as  human  tubercle 
bacilli  which  have  become  avirulent  by  prolonged  culture  on  artificial  media  (Krom- 
pechen).  Tuberculin  prepared  from  a  culture  of  an  ichthic  bacillus,  which  Ramond 
and  Ravaud  believe  to  be  the  same  as  the  tuberculin  obtained  from  a  culture  of  the 
human  bacillus,  does  not,  when  administered  in  ordinary  doses,  give  the  same 
reaction  as  Koch's  tuberculin  but  behaves  more  like  that  produced  from  a  culture 
of  an  avirulent  human  bacillus  (Krompecher). 


EXPERIMENTAL   INOCULATION  297 


Friedmann  has  recovered  from  two  cases  of  spontaneous  pulmonary  tuber- 
culosis in  tortoises  a  bacillus  which,  in  many  of  its  characteristics,  differs 
from  the  ichthic  bacillus  and  which  appears  to  be  intermediate  between  the 
ichthic  and  human  types  of  tubercle  bacilli.  Friedmann's  bacillus  grows 
both  at  ordinary  room-temperature  and  at  37°  C. :  it  is  not  pathogenic  to 
mammals  but  in  the  guinea-pig  sets  up  a  local  lesion  which  undergoes  spon- 
taneous resolution. 

4.  Organisms  associated  with  the  tubercle  bacillus. 

In  tuberculous  lesions  in  man  the  tubercle  bacillus  is  found  frequently  associated 
with  various  other  organisms,  the  latter  being  generally  of  a  pyogenic  nature.  In 
cavities  in  the  lungs  a  rich  microbial  flora  is  encountered  ;  the  following  among 
other  organisms  may  for  instance  be  found  in  the  lungs  in  conditions  of  human 
pulmonary  tuberculous  phthisis  :  staphylococci,  streptococci,  the  pneumobacillus  of 
Friedlander,  pneumococci,  bacillus  pyocyaneus,  micrococcus  tetragenus,  and  the 
bacteria  of  putrefaction.  The  hectic  fever  of  patients  suffering  from  tuberculosis 
is  due  to  the  absorption  of  toxins  secreted  by  these  micro-organisms  of  secondary 
infection.  In  glandular  and  meningeal  tuberculosis,  etc.  it  frequently  happens 
that  pneumococci,  streptococci,  and  staphylococci  are  found  together  with  the  tubercle 
bacillus. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

Guinea-pigs  or  rabbits  are  generally  inoculated  with  a  pure  culture  of  the 
bacillus  emulsified  in  a  little  sterile  water  [or,  better,  sterile  normal  saline 
solution]  or  with  tuberculous  tissues  pounded  in  a  mortar  with  a  few  drops 
of  water  [or  saline  solution] ;  or  the  material  (sputum,  pus,  small  pieces  of 
tissue,  etc.)  may  be  introduced  directly  either  beneath  the  skin  or,  in  the 
case  of  tissues,  into  the  peritoneal  cavity. 

A.  Guinea-pig. 

Guinea-pigs  inoculated  with  material  containing  even  a  few  tubercle  bacilli 
of  mammalian  or  avian  origin  invariably  become  infected  with  tuberculosis. 

[The  high  degree  of  virulence  of  the  avian  tubercle  bacillus  here  suggested 
was  not  confirmed  by  the  English  Commission.] 

Generally  speaking,  the  guinea-pig  is  less  susceptible  to  avian  than  to  human 
or  bovine  tubercle  bacilli.  According  to  Weber  and  Bofinger,  the  [sub-cutaneous] 
inoculation  of  an  avian  tubercle  bacillus  leads  to  a  localized  infection  in  guinea- 
pigs,  never  to  the  typical  disease.  [This  opinion  is  confirmed  by  the  English 
Commission  which  finds  that  "the  avian  bacillus  never  produces  a  progressive 
tuberculosis  in  the  guinea-pig."  ]  This  conclusion  however  is  not  supported  by  the 
work  of  numerous  other  observers. 

For  purposes  of  description  the  infection  set  up  by  the  inoculation  of  a 
mammalian  tubercle  bacillus  will  be  taken  as  a  type. 

1.  Sub-cutaneous  inoculation. — After  10  days  or  so  there  appears,  at  the 
site  of  inoculation,  a  small  indurated  nodule  which  later  softens  and  then 
forms  an  abscess  ;  this  abscess  opens  externally  leaving  an  ulcer,  the  so-called 
tuberculous  chancre.  At  the  same  time,  the  adjacent  glands  become  enlarged, 
the  animal  wastes,  becomes  cachectic  and  dies  in  from  1-3  months.  Post 
mortem  the  most  conspicuous  lesions  are  those  in  the  spleen  and  liver  :  the 
spleen  is  much  enlarged,  ochre-coloured,  speckled  with  caseous  tubercles  as 
well  as  with  more  recent  yellowish  granulations  ;  the  caseous  points  may 
have  become  confluent  giving  rise  to  irregular  whitish-yellow  mammillated 
masses  :  the  liver  shows  similar,  though,  as  a  rule,  less  extensive  lesions. 
The  surface  of  the  lungs  and  of  the  kidneys  and  the  serous  membranes  will 
be  found  covered  with  a  fine  sprinkling  of  miliary  granulations.  The  lym- 
phatic glands  in  the  neighbourhood  of  the  site  of  inoculation  are  caseous. 


298 


THE   TUBERCLE  BACILLUS 


If  the  animal  be  killed  within  a  fortnight  to  three  weeks  after  inoculation  the 
lesions,  especially  the  tubercles  on  the  spleen  and  liver,  will  be  found  to  have 
attained  their  characteristic  appearances.  At  this  period  of  the  disease  the 


Cervical  lymphatic  glands 


Post-sternal 
lymphatic  glands' 


Post-manubrial 
lymphatic  gland 


Axillary 
lymphatic  glands 


Inguinal 

lymphatic  glands  "~  - 


Axillary 
lymphatic  gU  n 


,  -  Mesenteric  gl;  n 


Inguinal 
~"  lymphatic  gla 


FIG.  189. — Tuberculous  guinea-pig  (sub-cutaneous  inoculation)  (3J  weeks). 

The  areas  marked  black  show  the  structures  mainly  affected,  viz.  the  inguinal, 
axillary,  post-manubrial,  post-sternal  and  cervical  lymphatic  glands  and  the 
spleen. 


lesions  are  most  marked  in  the  glands  on  the  same  side  as  and  adjacent  to 
the  site  of  inoculation.  It  is  only  towards  the  second  month  that  tubercles 
appear  in  the  bronchial  glands  and  lungs. 

These  appearances  were  first  described  by  Villemin  hence  this  type  of 
generalization  of  the  disease  is  sometimes  known  as  the  Villemin  type. 

[The  course  of  the  disease  in  guinea-pigs  following  the  sub-cutaneous  inocula- 
tion of  bovine  tubercle  bacilli  was  worked  out  by  A.  S.  and  F.  Griffith.     The 


INOCULATION   OF  GUINEA-PIGS  299 


material  was  inoculated  in  the  inguinal  region.  The  guinea-pig  killed  five  days 
later  showed  a  local  thickening  only.  The  ten-day  guinea-pig  showed  in 
addition  to  a  local  lesion,  lesions  in  the  superficial  inguinal  glands  and  in  the 
axillary  and  sternal  glands.  The  twenty-day  animal  showed  extension  of 
the  disease  to  the  deep  inguinal,  iliac  and  manubrial  glands  and  to  the  spleen, 
liver  and  portal  glands  :  one  tubercle  was  found  in  the  lung.  In  the  thirty- 
day  guinea-pig  the  disease  had  reached  the  lungs  and  bronchial  glands, 
the  intestines  and  mesenteric  glands  as  well  as  the  cervical,  lumbar  and 
coeliac  glands.  The  thirty-eight  day  guinea-pig  showed  tubercles  in  the 
kidneys. 

[The  duration  of  life  of  the  guinea-pig  will  depend  upon  the  dose  of  tubercle 
bacilli  administered  :  but  the  extent  of  the  disease  is  not  found  to  vary  much, 
since  an  extremely  small  number  of  either  bovine  or  human  tubercle  bacilli 
is  able  to  set  up  general  progressive  tuberculosis  in  the  guinea-pig  (English 
Commission).] 

2.  Cutaneous  inoculation.  —  If  the  inguinal  region  of  a  guinea-pig  be  shaved 
and  rubbed  with  a  piece  of  absorbent  wool  soaked  in  sputum  containing 
tubercle  bacilli,  the  corresponding  glands  will  become  enlarged  a  week  or 
fortnight  later  and  the  animal  will  die  of  tuberculosis  in  about  two  months. 
Post  mortem,  lesions  typical  of  the  disease  will  be  found  (Osman  Nouri). 
This  method  of  inoculation  is  very  useful  for  diagnosis,  because  it  involves 
no  risk  of  death  from  septicaemia,  an  accident  very  likely  to  happen  if  the 
material  be  inoculated  sub-cutaneously. 

3.  Intra-peritoneal  inoculation.  —  The  course  of  the  disease  is  similar  to 
that  just  described  but  is  more  rapid.     Death  occurs  in  2-6  weeks    being 
preceded  by  an  increasing  degree  of  cachexia.     Lesions  similar  to  those 
already    described    are    found    in    the 

tissues  :    the  peritoneum   is  infiltrated  ^  j»*W:»  "'*** 

with  tubercles  and  the  omentum  forms 

a    compact,    caseous    mass,    while    the 

mesenteric  and  inguinal  glands  are  also 

caseous.     The  indurated  nodule  at  the 

site  of  inoculation  (chancre)  is,  of  course, 

non-existent.  *        *       *        /B 

A  large  dose  of  an  human  or  avian  w  \  /$«•?  ^  .-^  \ 
culture  is  fatal  to  guinea-pigs  in  a  few  ^  i*  A  r~&\  V 
days  when  inoculated  intra-peritoneally.  (Ifc^  $  9 

Post  mortem  there  is  an  excess  of  fluid         v 
in  the  pleurae  but  no  tubercles  are  visible  ** 

in  the  internal  organs  (Koch,  Straus  and  *       A 

Gamaleia).  " 

[Following    intra-peritoneal    inocula- 
finn    flio    nrmrao    r>f     fTi»    rli«PQ«ap    i«    a«         FlG-  19°-  —  Scraping  from  the  spleen  of  a 

tion  tne  course  ol    tne   disease   is   as    tuberculous  gUinea-pig  (carboi-fuchsin    and 

follows  :    in    guinea-pigs    which    die    in     methylene  blue).     (Oc.  2,  obj.  Ath,  Zeiss.) 
under  14  days,  there  is  a  local  lesion 

in  the  wall  of  the  abdomen  :  the  omentum  is  thickened,  the  mesentery  and 
peritoneum  are  inflamed  and  covered  with  a  thin  membrane,  the  mesentery 
is  also  thickened  :  the  spleen  is  enlarged  and  speckled  with  minute  points, 
the  liver  shows  minute  foci,  the  kidneys  are  normal  :  the  pleural  cavities 
are  filled  with  fluid,  the  lungs  are  collapsed  and  often  consolidated  and  show 
minute  grey  points  in  the  dark  red  areas  :  the  pyloric,  lumbar  and  ventral 
mediastinal  (or  sternal)  glands  are  severely  affected,  the  portal  and  other 
abdominal  lymphatic  glands  less  affected  while  the  bronchial  glands  are 
usually  only  slightly  affected.  In  guinea-pigs  which  survive  for  3  weeks  to  a 


j  &  Si    «f%  2?  9 

£          .""^*';Y;. 

»  *  «     ^  f  ^p».  4. 


300  THE   TUBERCLE   BACILLUS 

month  there  is  severe  tuberculosis  of  the  peritoneum,  omentum  and  mesentery : 
the  spleen  is  enlarged  :  tubercles  are  visible  in  the  spleen,  liver  and  lungs  and 
sometimes  in  the  kidneys :  the  pleural  cavities  sometimes  contain  an  excess 
of  fluid  and  the  pleurae  are  covered  with  small  grey  tubercles.  "  With 
smaller  and  smaller  doses  of  tubercle  bacilli  the  lesions  in  the  organs  begin 
to  resemble  more  and  more  those  produced  by  sub-cutaneous  inoculation" 
(English  Commission).] 

4.  Intra-pulmonary  inoculation. — There  is  a  caseous  focus  at  the  point  of 
entry  of  the  inoculation  needle  and  the  lungs  are  spotted  with  grey  tubercles 
in  the  adjacent  area.     In  the  abdominal  viscera  the  lesions  are  similar  to  those 
following  sub-cutaneous  inoculation. 

5.  Inhalation. — Guinea-pigs  are  readily  infected  with  tuberculosis  by  causing 
them  to  inhale  dried  and  finely  powdered  tuberculous  sputum  or  dust  mixed 
with   tubercle   bacilli   from   cultures.     The   animal   dies   with   well-marked 
caseous  broncho-pneumonia. 

6.  Ingestion. — Guinea-pigs  have  been  infected  by  feeding  them  with  the 
milk  of  a  cow  suffering  from  tuberculous  phthisis  (Villemin  and  Parrot,  Klebs). 
In  animals  infected  in  this  way  it  occasionally  happens  that  there  are  no 
lesions  in  the  intestines.     [When  feeding  guinea-pigs  with  tubercle  bacilli 
Cobbett  sometimes  observed  a  generalized  infection  involving  the  lungs  but 
pulmonary  tuberculosis  apart  from  a  generalized  infection  did  not  occur.] 

B.  Rabbits. 

Rabbits  are  not  so  susceptible  to  tuberculosis  as  guinea-pigs.  A  fatal 
result  does  not  always  follow  the  inoculation  of  a  small  amount  of  tubercu- 
lous material.  Occasionally  the  local  lesion  is  of  long  standing  before  the 
disease  becomes  generalized.  Inoculation  of  bovine  or  avian  tubercle  bacilli 
is  followed  by  a  more  severe  infection  than  inoculation  with  human  tubercle 
bacilli. 

1.  Sub-cutaneous  inoculation. — According  to  the  amount  of  virus  inocu- 
lated the  animal  will  live  for  from  one  to  several  months.     A  local  induration 
(tuberculous  chancre)  is  formed  but  the  glands  are  often  not  affected  while 
the  lungs  are,  as  a  rule,  the  first  to  show  tubercles. 

The  human  tubercle  bacillus  often  fails  to  kill  rabbits  when  inoculated  sub-cutane- 
ously.  On  an  average,  four  out  of  five  strains  of  human  tubercle  bacilli  produce 
only  a  local  lesion  and  this  undergoes  spontaneous  resolution.  The  disease  resulting 
from  inoculation  of  bovine  bacilli  is  always  more  severe. 

2.  Intra-peritoneal  inoculation. — The  course  of  the  disease  is  more  rapid. 
Tubercles  are  found  on  the  peritoneum,  spleen,  liver  etc.     Death  often  occurs 
before  the  disease  has  reached  the  thorax. 

3.  Intra-pulmonary  inoculation.    Inhalation. — The  disease  runs  the  same 
course  and  presents  the  same  lesions  as  in  the  guinea-pig.     Frankel  and 
Troje  have  produced  caseous  pneumonia  in  rabbits  as  a  result  of  intra-tracheal 
inoculation. 

4.  Inoculation  into  the  anterior  chamber  of  the  eye. — By  inoculating  the 
bacillus  into  the  anterior  chamber  of  the  eye  the  progress  of  the  lesions  can 
be  easily  followed.     During  the  third  week,  the  iris  becomes  covered  with 
tuberculous  granulations,  the  eye  swells,  the  aqueous  humour  becomes  cloudy 
and  occasionally  the  whole  eye  is  transformed  into  a  purulent  abscess  :    the 
glands  of  the  neck  hypertrophy  and  infection  becomes  disseminated  giving 
rise  to  a  generalized  tuberculosis  of  the  Villemin  type  (p.  298). 

5.  Intra-venous    inoculation. — The    disease    produced    by    intra-venous 
inoculation  may  be  of  one  of  two  types  : 

(a)  Miliary  tuberculosis. — According  to  the  amount  of  material  inoculated 


INOCULATION  OF  RABBITS  301 

death  may  take  place  in  2  or  3  weeks.     The  viscera  and  serous  membranes 
are  covered  with  fine  miliary  tubercles. 


* 


FIG.  191. — The  figure  represents  the  lesion  produced  in  a  liver  of  a  rabbit 
3  days  after  inoculation  in  the  vein  of  an  ear  with  1  mg.  of  finely  emulsified 
culture  of  mammalian  tubercle  bacilli  and  illustrates  a  typical  tubercle,  with  a 
peripheral  infiltration  of  small  lymphocytes  and  finely  granular  oxyphil  leuco- 
cytes, x  600.  (Eastwood.)  (See  footnote  p.  295.) 

(b)  The  Yersin  type. — Death  takes  place  in  12-25  days.  The  animal  loses 
flesh  and  rapidly  becomes  cachectic.  The  temperature  is  very  much  raised. 
Post  mortem  the  only  visible  lesion  is  a  marked  hypertrophy  of  the  liver  and 
spleen.  There  are  no  visible  tubercles.  The  liver,  spleen  and  bone  marrow 
contain  enormous  numbers  of  bacilli. 

Strauss  and  Gamaleia  held  that  this  second  (Yersin)  type  of  infection  is  the 
characteristic  change  following  mtra-venous  inoculation  of  the  bacillus  of  avian 
origin,  but  numerous  facts  have  been  brought  forward  to  prove  that  intra-venous 
inoculation  of  the  bacillus  of  human  origin  may  result  in  this  type  of  infection  (Yersin, 
Nocard,  and  others).  Granchez  and  Ledoux-Lebard  have  produced  either  of  these 
two  types  of  infection  at  will  by  regulating  the  amount  of  material  inoculated. 

Generally  speaking,  however,  intra-venous  inoculation  of  the  rabbit  with  the 
avian  bacillus  produces  a  simple  tuberculous  infiltration  of  the  organs  without  any 
visible  lesions. 

[The  experiments  of  the  English  Commission  showed  that  when  death  occurred 
about  a  fortnight  after  the  inoculation  of  the  avian  tubercle  bacillus  into  the  veins, 
disease  of  the  Yersin  type  is  seen  post  mortem.  But  if  death  be  postponed  to  a  later 
period  tubercles  are  visible  in  the  internal  organs. 

[When  rabbits  were  inoculated  intra-venously  with  bovine  tubercle  bacilli  they 
sometimes  died  within  a  fortnight  of  a  very  acute  disease  which  did  not  altogether 
correspond  to  the  Yersin  type.  In  these  cases  the  lungs  were  solid  with  masses  of 
grey  tubercles,  the  bronchial  glands  were  cedematous  and  the  spleen  enlarged, 
there  were  indefinite  tuberculous  foci  in  the  liver  and  kidneys,  and  on  microscopical 
examination  tubercle  bacilli  were  found  to  be  numerous  in  all  the  tissues  of  the 
body. 

[The  intra-venous  inoculation  of  human  tubercle  bacilli  will  occasionally  lead  to 
an  acute  condition  similar  to  that  just  described  as  sometimes  following  intra- 
venous inoculation  of  bovine  tubercle  bacilli.  ] 


302 


THE   TUBERCLE   BACILLUS 


[The  results  of  the  inoculation  experiments  carried  out  on  rabbits  by 
Cobbett,  A.  S.  Griffith  and  F.  Griffith  on  behalf  of  the  English  Commission 
may  be  summarized  here. 

[The  bovine  tubercle  bacillus  produces  a  severe  and  fatal  general  tubercu- 
losis whether  inoculated  sub-cutaneously,  intra-venously  or  intra-peritoneally. 
[The  human  tubercle  bacillus  very  occasionally  produces  a  fatal  general 
tuberculosis  when  inoculated  intra-venously  or  intra-peritoneally  but  as  a  rule 
the  lesions  found  are  those  of  a  slight  and  retrogressive  tuberculosis.  Sub- 
cutaneous inoculation  never  leads  to  a  fatal  result :  for  example,  125  rabbits 
inoculated  sub-cutaneously  with  doses  varying  from  1-100  mg.  of  culture 
of  the  human  tubercle  bacillus  and  killed  after  3-24  months  all  showed 
retrogressive  tuberculosis. 

[The  avian  tubercle  bacillus  usually  produces  a  fatal  general  tuberculosis 
by  whichever  of  the  three  methods  it  be  inoculated.  This  type  is  less  virulent 

for  rabbits  than  the  bovine  tubercle 

£*  bacillus  and  it  causes  generally  less 

disease  of  the  internal  organs.  ] 

6.  Infection  by  feeding.— The  in- 
gestion  of  tuberculous  material 
mixed  with  food  does  not  always 
lead  to  infection  of  the  rabbit : 
some  animals  entirely  escape  the 
disease,  others  show  lesions  of  the 
alimentary  and  respiratory  tracts 
while  the  majority  contract  an 
infection  strictly  limited  to  the 
respiratory  passages  ( Weleminsky). 
The  sub -maxillary  glands  are  first 
infected  then  the  cervical  glands, 
followed  by  the  bronchial ;  finally 

the     pulmonary    parenchyma    is 

FIG.  192. — An  early  lesion  produced  in  the  liver  of  n4-4-nft~\r^J       rpr          "Ui^'4-  • 

a  rabbit  14  days  after  inoculation  in  the  vein  of  an  attacked.       Ine  rabbit  IS  more  SUS- 

ear  with  1  mg.  of  culture  of  avian  tubercle  bacilli,  ceptible  to  the  ingestion  of  bacilli 

An  example  of  a  "giant  cell"  produced  by  the  avian  -S                              .  &         .    .        , 

tubercle    bacillus.     The   bacilli  have   been    growing  OI    bovine   Or  avian  Origin  than  01 

abundantly  within  the  "cell"  and  are  very  small.  nr,Pilli  nf  Tinman  rni'm'n 

x  600.    (Eastwood.)    (See  footnote  p.  295.)  \  Ol  numan  origin. 

[Cobbett,  A.  S.  Griffith  and  F. 

Griffith  found  that  feeding  with  bovine  tubercle  bacilli  was  always  followed 
by  a  progressive  tuberculosis  in  rabbits  while  when  fed  with  the  avian 
tubercle  bacillus  only  one  rabbit  out  of  seventeen  fed  showed  severe  genera- 
lized tuberculosis.  Progressive  tuberculosis  cannot  be  produced  in  rabbits 
by  feeding  them  on  human  tubercle  bacilli.] 

C.  Dogs. 

Dogs  may  be  infected  by  inoculating  them  with  large  doses  of  the 
human  tubercle  bacillus  but  they  are  much  more  resistant  to  the  avian 
bacillus  though  not  absolutely  immune  to  it  (Grancher  and  Hericourt).  [The 
dog  is  one  of  the  few  species  of  animals  in  which  the  effects  produced  by  the 
bovine  and  human  tubercle  bacilli  are  identical.  Dogs  "  have  shown  them- 
selves insusceptible  to  avian  tubercle  bacilli  inoculated  by  the  most  severe 
method  and  in  relatively  large  doses  "  (work  of  the  English  Commission).] 

1.  Sub-cutaneous  inoculation. — The  disease  following  sub-cutaneous  inocu- 
lation is  not  necessarily  fatal :  it  may  remain  localized  or  become  a  generalized 
infection.  [The  English  Commission  found  that  the  dog  is  resistant  to  the 
sub-cutaneous  inoculation  of  either  the  bovine  or  the  human  tubercle  bacillus.] 


INOCULATION   OF  CATTLE 


303 


2.  Intra-peritoneal  inoculation.— Death  occurs  2-3  months  after  inocula- 
tion of  a  pure  culture  of  tubercle  bacilli  into  the  peritoneal  cavity.     Inoculation 
is  followed  by  peritonitis  with  excess  of  fluid,  the  formation  of  false  membranes, 
adhesion  of  the  coils  of  the  intestine,  and  infection  of  the  glands.     The  disease, 
ultimately,  becomes  generalized.     [Intraperitoneal  inoculation  with  moderate 
doses  of  cultures  of  either  the  human  or  the  bovine  tubercle  bacillus  is  usually 
but  not  invariably  fatal  (work  of  the  English  Commission).] 

3.  Intra- venous  inoculation. — Death  takes  place  1-2  months  after  inocula- 
tion into  a  vein  of  O25  c.c.  of  a  thick  emulsion  of  bacilli  from  a  glycerin-agar 
culture.     The  pulmonary  lesions  are  the  most  marked  while  the  liver,  spleen, 
etc.  may  also  show  tubercles. 

4.  Inhalation. — Tappeiner  infected  dogs  by  causing  them  to  breathe  an 
atmosphere  charged  with  dried  and  powdered  tuberculous  sputum.     Lesions 
were  found,  post  mortem,  in  the  lungs,  spleen  and  kidneys. 

5.  Infection  by  feeding. — Arloing  fed  dogs  with  cultures  of  the  tubercle 
bacillus  and  in  three  out  of  seven  cases  found  lesions  in  the  alimentary  canal ; 
in  two  other  cases  the  disease  was  generalized  (in  the  spleen  and  lungs). 
[Dogs  are  very  resistant  to  infection  with  tuberculosis  by  feeding,  especially 
adult  animals  (A.  S.  Griffith  and  F.  Griffith,  for  the  English  Commission).] 

D.  Cattle. 

(a)  Cattle  are  very  susceptible  to  infection  with  the  bacillus  of  the  bovine  type. 


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Fm.  193. — Section  of  the  udder  of  a  cow  which  died  299  days  after  intra- 
mammary  inoculation  of  bacilli  derived  from  a  typical  bovine  virus.  The 
specimen  is  taken  from  an  affected  portion  of  the  mammary  tissue  showing 
early  infection  Note — (1)  The  high  vascularity,  (2)  the  interstitial  infiltration 
with  bacilli,  (3)  the  entrance  of  bacilli  into  the  glandular  epithelium,  and  (4)  the 
excretion  of  bacilli  into  a  mammary  tubule.  It  is  of  importance  to  note  that 
lesions  such  as  this,  which  are  obviously  unrecognizable  clinically,  excrete 
numerous  bacilli.  Carbol-fuchsin  and  methylene  blue,  x  400.  (Eastwood.) 
(See  footnote  p.  295.) 

In  calves,  infection  by  way  of  the  alimentary  canal  leads  to  very  severe 
symptoms  (Vallee).     ["  Feeding  with  the  bovine  tubercle  bacillus  does  not 


304  THE   TUBERCLE   BACILLUS 

so  readily  set  up  general  progressive  tuberculosis  in  the  calf  as  does  inocula- 
tion "  (English  Commission).]  Feeding  is  the  most  certain  method  of 
infecting  the  tracheal  and  bronchial  glands  (Vallee,  Calmette  and  Guerin). 
The  bacilli  may  pass  through  the  intestinal  wall  without  producing  any 
apparent  lesion  either  of  the  mucous  membrane  or  of  the  mesenteric  glands 
provided  that  very  small  doses  of  bacilli  and  young  animals  be  used,  condi- 
tions, that  is,  similar  to  those  obtaining  in  the  spontaneously  contracted 
disease  (Vallee). 

Calmette,  Guerin  and  Delearde  fed  calves  with  O'l  gram  of  bovine  bacilli  and 
found  that  they  reacted  to  tuberculin  45  days  later.  The  tracheal  and  bronchial 
glands  were  swollen  and  hard  but  not  caseous,  the  mesenteric  glands  were  normal  in 
appearance,  but,  on  inoculation,  both  sets  infected  guinea-pigs. 

(b)  Cattle  can  also  be  infected  with  some  strains  of  bacilli  of  human  origin 
(Chauveau,  Ravenel,  Arloing,  M.  Wolff,  Schottelius,  Spronck  and  others). 
[These  strains  were  no  doubt  strains  of  bovine  tubercle  bacilli  infecting 
human  tissues  (vide  ante).  The  English  Commission  has  demonstrated  that 
the  human  tubercle  bacillus  is  incapable  of  causing  progressive  tuberculosis 
in  bovine  animals.] 

Schottelius  fed  bovine  animals  on  several  occasions  with  tuberculous  sputum. 
In  cows  he  found  a  tuberculous  enteritis  with  caseous  glands  ;  and,  in  calves, 
caseation  of  the  sub-maxillary  and  mesenteric  glands.  [This  should  be  read 
in  conjunction  with  the  comment  above.  The  English  Commission  investi- 
gated two  cases  of  pulmonary  tuberculosis  in  which  the  sole  infecting  agent 
was  the  bovine  tubercle  bacillus.]  In  calves,  bacilli  from  human  lesions 
whether  inoculated  beneath  the  skin  or  into  the  lungs  or  veins  produced 
general  tuberculosis  (De  Jong,  Sturmann).  Inoculation  (sub-cutaneous  and 
intra-peritoneal)  of  human  tuberculous  material  into  calves  may  lead  to  a 
rapid  and  generalized  infection  (Fibiger  and  Jensen,  Eber)  [if  the  bacilli  of 
human  origin  are  of  the  bovine  type  not  otherwise  (English  Commission)]. 

In  two  cases  in  which  Eber  obtained  a  very  severe  infection  in  calves  the  bacilli 
were  derived  from  children  in  whom  only  intestinal  lesions  were  present.  It  may 
be  admitted  that  the  children  were  infected  by  swallowing  bovine  bacilli  but  it  is  no 
less  true  that  human  tuberculosis  can  infect  calves  and  bovine  tuberculosis  children. 
[It  seems  to  be  a  perfectly  justifiable  inference  from  the  work  of  the  English  Com- 
mission that  the  bacilli  used  by  Eber  in  which  he  produced  a  severe  infection  in 
calves  must  have  been  derived  from  children  suffering  from  an  infection  with  bovine 
bacilli.  Tubercle  bacilli  of  the  human  type  merely  give  rise  to  a  slight  and  retro- 
gressive type  of  tuberculosis  in  calves  and  in  the  sense  that  human  tuberculosis 
due  to  the  bovine  tubercle  bacillus  can  infect  calves  the  statement  in  the  preceding 
paragraph  is  true.  With  regard  to  the  reciprocal  infection  of  children  by  bovine 
tuberculosis  it  may  be  pointed  out  that  fourteen  out  of  the  twenty-seven  cases  of  ali- 
mentary tuberculosis  investigated  by  the  English  Commission  were  due  to  bovine 
tubercle  bacilli.]  Moreover,  Eber  produced  an  acute  miliary  tuberculosis  with 
tuberculous  material  from  an  adult  human  being  suffering  from  pulmonary  tubercu- 
losis and  tuberculous  meningitis.  [Probably  an  infection  produced  by  the  bovine 
tubercle  bacillus.  See  English  Commission  results,  ante.] 

Such  facts  [may  be  considered  to]  constitute  a  sufficient  basis  for  rejecting  Koch's 
hypothesis  of  the  existence  of  two  separate  and  distinct  species  of  tubercle  bacilli. 

E.  Birds. 

(a)  Birds  are  easily  infected  with  the  avian  tubercle  bacillus.  Fowls  may 
be  infected  by  any  method  of  inoculation  (sub-cutaneous,  intra- venous, 
feeding  etc.),  and  the  ingestion  of  cultures,  infected  tissues  or  other  patho- 
logical tuberculous  products  readily  produces  the  disease.  Post  mortem 
tubercles  are  found  on  the  abdominal  viscera  but  chiefly  in  the  [spleen  and] 
liver. 


INOCULATION   OF  BIRDS  305 

Intra-venous  inoculation  of  the  avian  bacillus  leads  to  the  death  of  the 
fowl  in  a  fortnight  to  three  weeks  with  a  disease  of  the  Yersin  type  (vide  also 
p.  301).  [In  the  experience  of  the  English  Commission  it  was  generally  longer 
— 5  to  6  weeks.] 

(6)  Those  who  believe  that  the  human  and  avian  tubercle  bacilli  belong  to 
different  species  hold  that  the  fowl  cannot  be  infected  with  the  human  tubercle 
bacillus  :  but  this  conclusion  [in  the  opinion  of  many]  can  no  longer  be 
maintained  in  view  of  the  experiments  carried  out  by  Koch,  Nocard  and 
Cadiot,  and  Gilbert  and  Roger.  These  observers  [appear  to]  have  shown 
that  the  fowl  becomes  infected  with  tuberculosis  as  the  result  of  the  ingestion 
of  human  tuberculous  material  and  of  pure  cultures  of  human  tubercle  bacilli. 
It  is  [by  some  considered  as]  certain  that  the  fowl  may  become  infected  by 
the  ingestion  of  the  sputum  of  phthisical  persons. 

It  has  to  be  remembered  that  fowls  often  resist  infection  with  bacilli  of 
human  origin  and  that  when  infection  does  occur  the  disease  is  chronic  and 
leads  to  the  formation  of  tubercles  in  the  internal  organs.  [Fowls  are  resistant 
to  mammalian  tubercle  bacilli  of  whatever  source  when  inoculated  intra- 
peritoneally,  sub-cutaneously  and  by  feeding,  but  frequently  succumb  to  intra- 
venous inoculations.  Tubercle  bacilli  killed  by  exposure  to  steam  at  100°  C., 
whether  avian  or  mammalian,  may  produce  however  •  similar  effects  when 
inoculated  intra-venously  into  the  fowl ;  these  effects  are  therefore  not  a  true 
tuberculosis  but  are  to  be  attributed  to  the  toxic  action  of  the  bacilli  (F. 
Griffith,  for  the  English  Commission).] 

F.  Cold-blooded  animals. 

Frogs  and  fish  do  not  appear  capable  of  infection  with  bacilli  of  human  and 
avian  origin.  But  there  is  an  observation  to  the  effect  that  true  tubercles 
have  been  produced  by  inoculating  bacilli  of  human  origin  into  the  peritoneal 
cavity  of  frogs  and  carp  (Moret). 

Bertarelli  [is  stated  to  have]  succeeded  in  infecting  snakes  (Varanus  varius) 
by  inoculating  them  under  the  skin  with  human  tuberculous  sputum  but 
failed  with  cultures  of  bacilli  of  avian  origin. 

Moeller  infected  the  blind  worm  [Anguis  fragilis]  with  bacilli  of  human 
origin  (p.  334). 

Sorgo  and  Suess  produced  tuberculous  lesions  (caseating  masses  [at  the 
site  of  inoculation]  and  occasionally  generalization)  in  two  blind  worms  and 
four  snakes  with  bacilli  of  human  origin,  though  many  of  their  experiments 
were  negative.  In  blind  worms  the  bacilli  retain  all  the  characteristics  associ- 
ated with  the  human  tubercle  bacillus  but  in  snakes  they  [are  said  to  ]  undergo 
a  partial  change  and  to  develop  some  of  the  characteristics  of  bacilli  of  ichthic 
origin. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

Human,  avian  and  ichthic  tubercle  bacilli  all  have,  in  the  main,  the  same 
characteristics.  In  cultures  they  are  small,  very  slender,  generally  non- 
motile,  rods. 

Ferran  says  that  the  tubercle  bacillus  is  motile,  but  the  conclusions  arrived  at 
in  his  paper  cannot  all  be  accepted  unreservedly.  Arloing  confirms  Ferran's  opinion. 
By  sub -cultivating  a  glycerin-potato  culture  on  to  glycerin- broth  this  observer 
obtained  motile  bacilli.  Schumowsky,  in  a  similar  experiment,  also  found  motile 
bacilli.  Auclair  [is  said  to  have]  succeeded  in  converting  the  tubercle  bacillus  into 
a  motile  saprophyte,  etc. 

In  cultures  on  solid  media  the  bacilli  are  arranged  in  long  wavy  coils  some- 

u 


306 


THE  TUBERCLE   BACILLUS 


thing  like  a  moustache  due  to  the  regular  interlocking  of  the  bacilli  with  a 
common  orientation. 

This  arrangement  of  the  bacilli  can  be  readily  shown  by  lightly  pressing  a  cover- 
glass  on  the  surface  of  a  glycerin-agar  culture  and  lifting  it  off  without  friction. 
The  film  should  be  fixed  by  heat  and  stained  by  one  or  other  of  the  methods  described 
below.  On  examining  with  an  oil-immersion  lens  the  appearances  reproduced  in 
fig.  194  will  be  seen. 


FIG.  194.  —  Tubercle  bacillus  :  impression  preparation.     (After  Koch.) 


The  bacillus  must  be  stained  before  it  can  be  found  in  fluids  and  tissues 
and  it  will  be  necessary  to  describe  the  various  methods  of  staining  before 
embarking  upon  a  description  of  its  characteristics. 

Staining  methods. 

Special  methods  have  to  be  adopted  in  order  to  stain  the  tubercle  bacillus. 

The  tubercle  bacillus  is  difficult  to  stain  with  the  basic  aniline  dyes  but  once 
stained  it  resists  the  decolourizing  action  of  such  powerful  agents  as  dilute  mineral 
acids.  Only  two  other  pathogenic  bacilli  share  this  characteristic  with  the 
tubercle  bacillus  viz.  the  leprosy  bacillus  from  which  it  is  easily  distinguished 
and  the  bacillus  of  Verruga  peruana.1 

This  acid-fast  property  of  the  tubercle  bacillus  serves  as  a  means  of  diagnosing 
the  organism  in  fluids  and  tissues  in  which  it  is  present.  The  property  of  resisting 
the  decolourizing  action  of  acids  seems  to  be  due  to  the  presence  of  a  fatty  or  waxy 
substance  insoluble  in  alcohol  and  ether  (Koch,  Tavel,  Viquerat).  By  treating  the 
bacilli  with  warm  xylol,  Borrel  extracted  a  waxy  substance  which  was  acid-fast 
while  the  bacilli  had  lost  this  property.2 

1  Besides  these  two  there  are  a  few  other  bacilli,  like  the  tubercle  bacillus,  capable 
when  deeply  stained  of  resisting  the  decolourizing  action  of  dilute    acids.     Such,  for 
instance,  are  the  smegma  bacillus  and  the'  bacillus  of  Tavel  (the  so-called  syphilis  bacillus 
of  Lustgarten)  —  but  these,  unlike  the  tubercle  bacillus,   are  decolourized  by  absolute 
alcohol  or  ether  —  likewise  the  various  acid-fast  bacilli  of  Bienstock,  Gottstein,  Moller, 
Rabinowitsch,  etc.     (Vide  infra,  The  acid-fast  bacilli.} 

2  In  opposition  to  the  opinion  expressed  by  H.  Aronson,  Sabrazes  has  shown  that  by 
treating  tissues  for  sections  with  ether,  xylol'and  chloroform  the  characteristic  staining 
properties  of  the  tubercle  bacillus  are  in  no  way  interfered  with.     And  the  same  is  true 
of  picric  acid,  carbolic  acid  and  perchloride  of  mercury  none  of  which  prevent  subse- 
quent staining  by  the  Ziehl-Neelsen  method.     On  the  other  hand,  undiluted  mineral 
acids,  2  per  cent,  chromic  acid,  formalin,  bichromates,  osmic  acid,  etc.  either  interfere  with 
or  entirely  prevent    subsequent   staining  by  the   carbol-fuchsin   method.     [Eastwood, 
however,  working  for  the  English  Commission  hardened  tissues  in  formalin  (p.  338).] 


MORPHOLOGY  307 

Numerous  methods  of  staining  the  tubercle  bacillus  have  been  suggested, 
but  they  all  depend  upon  the  principle  enunciated  above. 

The  various  methods  in  most  frequent  use  will  now  be  described,  but  the  necessity 
for  beginners  to  limit  themselves  to  one  method  which  they  thoroughly  understand 
and  upon  the  results  of  which  they  can  rely  cannot  be  too  strongly  emphasized. 
The  Ziehl-Neelsen  method  is  by  far  the  best. 

The  tubercle  bacillus  stains  by  Gram's  method  but  with  difficulty  and  the 
stain,  carbol-  or  anilin-gentian-violet  (Nicolle),  must  be  allowed  to  act  for,  at 
least,  10  minutes.  The  bacilli  are  always  granular  by  this  method. 

Much  has  shown  that  in  cattle  inoculated  with  tuberculosis,  tuberculous  nodules 
are  often  seen,  post  mortem,  in  the  lungs  in  which  no  bacilli  can  be  demonstrated  by 
Ziehl's  method,  though  the  presence  of  bacilli  in  the  lesions  is  proved  by  the  result 
of  inoculations  (the  same  is  true  of  "  cold  abscesses  "  in  man).  If,  however,  Gram's 
method  of  staining  be  adopted,  leaving  the  preparations  in  the  violet  for  48  hours 
and  in  the  iodine  solution  for  24  hours,  large  numbers  of  bacilli  can  be  seen  in  the 
lesions.  From  this  observation  Much  concludes  that  in  addition  to  the  acid-fast 
tubercle  bacillus  there  is  a  virulent  form  which  is  non-acid-fast. 

A.  The  staining  of  films. 
1.  Ziehl-Neelsen  method. 

Method  recommended. 

The  principle  of  the  method. — If  a  film  stained  with  carbol-fuchsin  be 
treated  with  a  diluted  mineral  acid,  the  background  and  all  the  organisms, 
with  the  exception  of  the  tubercle  bacillus  (and  also  those  of  leprosy,  verruga 
and  the  "  acid-fast  "  bacilli,  pp.  350  and  345),  will  be  decolourized.  The 
tubercle  bacillus  retains  its  red  colour.  If,  now,  the  preparation  be  stained  with 
an  aqueous  solution  of  methylene  blue,  the  background  and  the  decolourized 
organisms  take  up  the  blue  while  the  tubercle  bacillus  remains  red. 

Technique. — 1.  Spread,  dry  and  fix  a  film  on  a  cover-glass  in  the  ordinary 
way.  Hold  it  in  a  pair  of  Cornet's  forceps  and  flood  it  with  a  large  drop  of 


FIG.  195.  —  Tubercle  bacilli  in  sputum.    Carbol-fuchsin  and  methylene  blue. 


(Oc.  2,  obj.  jUh,  Zeiss.) 


Ziehl's  carbol-fuchsin  (p.  138).  Hold  the  cover-glass  over  a  small  flame  (the 
pilot  light  of  a  Bunsen,  for  example)  and  heat  very  gently  until  steam  just 
begins  to  rise  ;  continue  the  heating  for  two  minutes,  being  careful  not  to 
boil  the  stain  and  to  see  that  the  staining  solution  does  not  dry  up. 


308  THE  TUBERCLE   BACILLUS 

2.  Pour  off  the  stain  and  treat  for  a  few  seconds  with  a  33  per  cent,  solution 
of  nitric  acid  (distilled  water  2  volumes,  pure  nitric  acid  1  volume)  or  a  25  per 
cent,  solution  of  sulphuric  acid  (distilled  water,  3  volumes,  pure  sulphuric 
acid,  1  volume),  [or  25  per  cent,  hydrochloric  acid  (Eastwood)].     The  pre- 
paration now  assumes  a  yellowish  tint. 

[This  method  of  decolourization  appears  to  be  perfectly  satisfactory  in  the  case 
of  bacilli  from  cultures  and  was  moreover  the  method  adopted  by  Eastwood  in  his 
work  for  the  English  Commission.  But  it  is  undoubtedly  true  that  tubercle  bacilli 
direct  from  human  and  animal  tissues — in  sputum,  for  example — will  sometimes 
lose  the  stain  in  these  strong  acids.  In  searching  for  the  tubercle  bacillus  therefore 
in  fresh  material  in  which  its  presence  is  suspected  it  is  recommended  that  a  2 '5  per 
cent,  solution  of  sulphuric  or  hydrochloric  acid  be  used  and  that  the  film  be  not 
exposed  to  the  acid  for  a  longer  time  than  is  absolutely  necessary.  ] 

3.  Wash  freely  in  water  :  the  preparation  should  now  be  pale  pink,    and  if 
the  pale  pink  colour  does  not  appear,  the  decolourization  has  been  insufficient 
and  the  film  must  be  treated  with  acid  again. 

4.  Pour  a  few  drops  of  absolute  alcohol  on  the  film  :   when  decolourization 
is  complete  the  film  should  be  a  very  faint  pink  colour. 

By  using  alcohol  decolourization  can  be  pushed  much  further  than  would  be  possible 
with  acid  since  the  latter  would  ultimately  decolourize  the  tubercle  bacillus.  A 
further  advantage  in  using  alcohol  is  that  it  decolourizes  the  smegma  bacillus  and 
thus  a  possible  source  of  error  is  eliminated. 

5.  Wash  well  in  water.     Stain  for  a  few  moments  with  an  aqueous  solution 
of  methylene  blue. 

6.  Wash  in  water.     Dry.     Mount  in  balsam. 

Note. — When  it  is  merely  a  question  of  searching  for  the  tubercle  bacillus  it  is  a 
great  advantage  not  to  counterstain  the  background  after  decolourizing  with  alcohol ; 
the  tubercle  bacilli  are  much  more  easily  seen  when  they  appear  stained  deep  red 
on  an  unstained  or  faintly  pink  background. 

For  this  purpose  the  above  procedure  is  stopped  at  the  end  of  Stage  4  and,  after 
washing,  the  preparation  is  examined  in  water.  If  after  examination  it  be  thought 
desirable  to  keep  the  film  it  may  be  counterstained  with  blue  and  treated  as  described 
above. 

This  simpler  method  is  particularly  applicable  when  the  bacilli  are  likely  to  be 
present  in  small  numbers  ;  in  any  case  it  renders  the  detection  of  the  bacilli  more 
rapid,  and  beginners  will  find  it  of  great  use. 

2.  Ehrlich's  method. 

1.  Stain  the  film  for  5  minutes  in  the  warm  with  aniline- violet. 

2.  Decolourize  in  33  per  cent,  nitric  acid  for  a  few  seconds. 

3.  Wash  in  water  :    continue  the  decolourization  with  absolute  alcohol. 

4.  Stain  for  a  few  seconds  in  the  cold  in  a  saturated  aqueous  solution  of 
vesuvin. 

5.  Wash.     Dry.     Mount. 

The  tubercle  bacilli  are  stained  violet,  other  structures  brown. 

3.  Gabbe's  method. 

This  method  is  merely  a  modification  of  Ziehl's  but  is  less  reliable  and  more 
difficult. 

1.  Stain  with  carbol-fuchsin  as  above. 

2.  Decolourize  and  counterstain  at  the  same  time  by  dipping  the  film  for  a  minute 
in  the  following  solution. 

Methylene  blue,         -         -  2  grams. 

25  per  cent,  sulphuric  acid,         -  -         100  c.c. 

3.  Wash.     Dry.     Mount. 

The  methods  described  by  Stocquart,  by  Pithion  and  Roux  (of  Lyon)  are  modifica- 
tions of  the  above  but  are  of  no  interest. 


MORPHOLOGY  309 

4.  Spengler's  method. 

1.  Stain  the  films  by  gently  warming  them  in  Ziehl's  solution. 

2.  Treat  for  a  few  seconds  with  picric-alcohol. 

Saturated  aqueous  solution  of  picric  acid,    -          -          -  60  c.c. 

95  per  cent,  alcohol,  40     ,, 

3.  Wash  three  times  in  60  per  cent,  alcohol. 

4.  Decolourize  rapidly  (about  20  seconds)  in  1  in  6  nitric  acid,  then  in  60  per  cent, 
alcohol. 

5.  Treat  again  with  picric-alcohol.     Wash.     Dry.     Mount. 

5.  Frankel's  method. 

1.  Stain  the  films  in  the  warm  for  5  minutes  with  aniline-fuchsin  (prepared  in  the 
same  way  as  aniline-violet  using  an  alcoholic  solution  of  fuchsin  instead  of  alcoholic 
gentian- violet). 

2.  Transfer  the  films  direct  to  the  following  solution  for  1  minute. 

90  per  cent,  alcohol,  50  c.c. 

Aniline  water.  30     ., 

Pure  nitric  acid,         -  20     ,, 

Saturated  alcoholic  solution  of  methylene  blue,  -  Q.S.  to  obtain  a 

deep  blue  colour. 

3.  Wash  in  distilled  water.     Dry.     Mount. 

6.  Herman's  method. 
Prepare  the  following  solutions  : 

A.  Krystal  violet,  1  gram. 
95  per  cent,  alcohol,                                                                                     30  c.c. 

B.  Ammonium  carbonate,  -  1  gram. 
Distilled  water,                                                                               -         100  c.c. 

Immediately  before  use,  pour  three  parts  of  solution  B  into  a  watch-glass,  add 
one  part  of  solution  A  and  mix  intimately. 

1.  Heat  the  staining  bath  until  it  just  begins  to  boil  and  leave  the  films  in  it  for 
a  minute. 

2.  Transfer  the  films  to  a  10  per  cent,  solution  of  nitric  acid  for  4  or  5  seconds. 

3.  Wash  in  absolute  alcohol  to  complete  the  decolourization. 

i> 


FIG.  196. — Tubercle  bacilli  in  sputum  :  Herman's  method. 
(Oc.  2,  obj.  Ath,  Zeiss.) 

4.  Transfer  the  films  for  30  seconds  to  the  following  solution  : 

Eosin,      -  1  gram. 

60  per  cent,  alcohol, 

5.  Wash  very  rapidly  in  alcohol.     Dry.     Mount.     The  tubercle  bacilli  are  stained 
violet  and  the  background  bright  pink. 


310  THE   TUBERCLE   BACILLUS 


7.  Lustgarten's  method  (modified). 

Sabouraud,  by  slightly  modifying  the  method  devised  by  Lustgarten  for  staining 
his  so-called  bacillus  of  syphilis,  has  perfected  a  method  of  staining  the  tubercle 
bacillus  which  he  affirms  to  be  very  delicate  and  precise.  The  method  is  as  follows  : 

1-  Stain  the  film  in  Ziehl's  solution  in  the  cold  for  1  or  2  hours  or  at  50°  C.  for  15 
minutes. 

2.  Treat  the  film  for  1-3  seconds  with  a  1  *5  per  cent,  solution  of  potassium  per- 
manganate. 

3.  Dip  at  once  into  a  freshly  prepared,  saturated,  aqueous  solution  of  sulphurous 
acid  for  a  few  seconds  until  the  film  is  decolourized. 

The  sulphurous  acid  solution  can  be  conveniently  prepared  by  bubbling  the  gas 
from  a  cylinder  of  liquefied  sulphurous  acid  through  distilled  water. 

4.  Wash  in  water  and  counterstain  with  an  aqueous  solution  of  methylene  blue 
for  1-3  minutes. 

5.  Wash  in  water.     Dry.     Mount  in  balsam. 

8.  Koch's  method. 

This,  the  earliest  method  used  for  the  detection  of  the  tubercle  bacillus,  is  chiefly  of 
historical  interest. 

1.  Place  the  films  for  1  day  at  room  temperature  or  for  a  few  hours  at  45°-50°  C. 
in  the  following  bath  : 

Saturated  alcoholic  solution  of  methylene  blue,    -  1  c.c. 

10  per  cent,  aqueous  solution  of  potash,       -  2     „ 

DistiUed  water,  -         200     „ 

2.  Transfer  the  films  to  a  saturated  aqueous  solution  of  vesuvin ;   in  about  a 
quarter  of  an  hour  a  brown  tint  takes  the  place  of  the  original  blue  colour  save 
in  the  tubercle  bacilli  which  still  retain  the  blue  stain. 

B.  The  staining  of  sections. 

The  methods  just  described  are  applicable  with  slight  modification  to  the 
staining  of  sections  :  but  in  this  case  it  is  essential  that  the  staining  should 
always  be  done  in  the  cold. 

1.  Ziehl-Neelsen's  method. 
Method  recommended. 

1.  Stain  the  section  in  the  cold  for  15-30  minutes  in  Ziehl's  fuchsin. 

2.  Decolourize  in  the  acid  solution  for  a  few  seconds.     Wash  in  water. 

3.  Continue  the  decolourization  with  absolute  alcohol  until  the  section  is 
a  pale  pink  colour.     Wash  in  water. 

4.  Stain  the  groundwork  with  an  aqueous  solution  of  methylene  blue. 

5.  Wash.     Pass  rapidly  through  absolute  alcohol,  clove  oil,  and  xylol. 
Mount  in  balsam. 

2.  Kiihne's  method. 
Method  recommended. 

The  following  unpublished  method  of  Kuhne  has  been  quoted  by  Borrel. 
It  is  particularly  useful  for  staining  sections  of  lung.  The  action  of  the 
hydrochloride  of  aniline,  which  is  the  decolourizing  agent  used,  is  not  so  rough 
as  that  of  mineral  acids  and  does  not  alter  the  arrangement  and  shape  of 
the  cells. 

1.  Stain  the  section  for  2  minutes  in  Bcehmer's  hsematoxylin  or  hsematein 
(p.  218)  to  stain  the  nuclei  of  the  cells.     Wash  in  distilled  water. 

2.  Stain  in  the  cold  with  Ziehl's  fuchsin  for  a  quarter  of  an  hour. 

3.  Decolourize  for  30-60  seconds  in  a  2  per  cent,  aqueous  solution  of  aniline 
hydrochloride. 

4.  Continue  the  decolourization  with  absolute  alcohol. 

The  cells  of  the  groundwork  are  now  unstained  with  the  exception  of  the  nuclei. 


MORPHOLOGY  311 


The  section  may  be  treated  with  orange-yellow  which  stains  particularly  the  red 
cells  of  the  blood.     After  staining  with  orange,  dehydrate  in  absolute  alcohol. 
5.  Clear  in  clove  oil  and  xylol.     Mount  in  balsam. 


WM 


<3R ,, 


FIG.  197. — Section,  of  human  tuberculous  lung.     Carbol-fuchsin  and  methy- 
lene  blue.    (Oc.  2,  obj.  /2th,  Zeiss.) 

3.  Ehrlich's  method. 

1.  Stain  in  aniline-violet  for  12  hours  in  the  cold. 

2.  Decolourize  in  33  per  cent,  nitric  acid  for  a  few  seconds.     Wash. 

3.  Complete  the  decolonization  in  absolute  alcohol. 

4.  Counterstain  with  a  saturated  aqueous  solution  of  vesuvin. 

5.  Dehydrate  rapidly  in  absolute  alcohol.     Clear  with  clove  oil  and  xylol. 
Mount  in  balsam. 

4.  Letulle's  method. 

1.  Stain  the  nuclei  with  haematoxylin  as  in  Kiihne's  method.     Wash  in  distilled 
water. 

2.  Stain  with  Ziehl's  fuchsin  in  the  cold  for  a  quarter  of  an  hour.     Wash  rapidly 
in  distilled  water. 

3.  Wash  in  absolute  alcohol  for  30  seconds. 

4.  Treat  with  the  following  solution  for  5  minutes  : 

lodgriin,  -----  1  gram. 

2  per  cent,  solution  of  carbolic  acid,    -  -         100  c.c. 

5.  Decolourize  in  absolute  alcohol. 

6.  Clear  in  clove  oil  and  xylol.     Mount  in  balsam. 

The  groundwork  is  stained  very  pale  grey-lilac ;  the  nuclei,  violet ;  the  bacilli, 
deep  red.  This  method  can  be  used  for  tissues  hardened  in  Mailer's  fluid. 

5.  Herman's  method. 

Herman's  method  (p.  309)  can,  according  to  its  author,  be  applied  to  the  staining 
of  frozen  sections  of  tissues  fixed  in  acetic  perchloride  solution.  The  technique  is 
the  same  as  for  films,  the  stain  being  allowed  to  act  for  1  minute  with  steam  rising. 

6.  Lustgarten's  method  (modified). 

1.  Stain  for  some  hours  in  the  cold  in  carbol-fuchsin. 

2,  3,  4,  5.  As  for  films  (p.  310). 

6.  Wash  in  water.     Dehydrate  rapidly  in  absolute  alcohol. 

7.  Clear  in  clove  oil  and  xylol.     Mount  in  balsam. 

This  method  is  useful  when  searching  for  bacilli  in  sections  of  the  liver,  where 
they  are  often  difficult  to  find.  It  is  also  available  for  tissues  hardened  in  Miiller's 
fluid. 


312 


THE   TUBERCLE   BACILLUS 


Appearance  of  the  bacilli  in  stained  preparations. 

In  stained  preparations,  tubercle  bacilli  vary  in  length  from  2-5/A  and  in 
breadth  from  03-0'5/x.  They  are  generally  of  the  same  thickness  throughout. 
In  some  preparations  the  bacilli  are  homogeneous,  while  in  others  they  appear 
as  though  composed  of  a  number  of  small  oval  or  rounded  grains  separated 


FIG.  198. 


FIG.  199. 


FIG.  198. — From  a  culture,  20  days  old,  on  inspissated  horse  serum,  of  a 
mammalian  virus  of  low  experimental  pathogenicity  to  bovines  and  rabbits 
and  of  vigorous  cultural  growth.  The  virus  was  isolated  from  a  human  being. 
The  bacilli  are  very  short ;  some  of  them  show  a  central  constriction.  The 
clump  at  the  bottom  of  the  figure  illustrates  the  tendency  of  mammalian  bacilli 
to  stick  together  and  the  difficulty  of  separating  them  by  emulsification.  The 
bacilli  from  serum  cultures  of  this  virus  proved  shorter  than  the  average  vigor- 
ously growing  bacillus  of  human  origin.  This  figure  illustrates  the  impossibility 
of  distinguishing  with  certainty,  under  the  microscope,  "  human "  from 
"  bovine "  bacilli.  Carbol-fuchsin.  x  2150.  (Eastwood.)  (See  footnote 
p.  295.) 

FIG.  199. — From  a  culture,  22  days  old,  on  glycerinated  broth,  of  a  typical 
mammalian  virus  of  low  pathogenicity  to  experimental  animals  and  of  vigorous 
cultural  growth.  The  virus  was  isolated  from  a  human  being.  The  bacilli, 
obtained  from  a  copious  growth,  are  for  the  most  part  long  and  curved,  and 
with  a  tendency  to  beading.  Carbol-fuchsin.  x  2150.  (Eastwood.)  (See 
footnote  p.  295.) 


FIG.  200. 


FIG. 201. 


FIG.  200. — From  a  culture,  44  days  old,  on  glycerin-agar,  of  a  mammalian 
virus  which  was  isolated  from  the  bronchial  gland  of  a  human  being.  The 
occurrence  of  branched  forms  of  the  mammalian  tubercle  bacillus  in  cultures 
obtained  from  ordin.ary  media,  such  as  glycerinated  agar,  broth,  or  potato,  is, 
in  Eastwood's  experience,  extremely  rare.  The  figure  also  shows  other  forms 
of  bacilli,  some  very  long,  some  very  short,  and  many  with  globular  or  oval, 
darkly  stained  bodies  variously  situated.  Bacilli  with  such  appearances  as  these 
are  common  in  agar  cultures.  Carbol-fuchsin.  x  2150.  (Eastwood.)  (See 
footnote  p.  295.) 

FIG.  201. — Tubercle  bacilli  from  milk  obtained  from  the  udder  of  a  cow, 
which  had  received  an  intra-mammary  inoculation  with  a  typical  bovine 
virus.  The  bacilli  are  of  various  lengths  ;  many  of  them  are  curved  and 
regularly  beaded.  The  bacilli  here  shown  are  such  as  are  commonly  met  with 
in  cow's  milk  ;  it  would  obviously  be  impossible  for  anyone  to  decide,  on 
morphological  grounds,  that  they  were  of  bovine  rather  than  of  human  origin. 
Carbol-fuchsin.  x  2150.  (Eastwood.)  (See  footnote  p.  295.) 


MORPHOLOGY 


313 


by  clear  unstained  intervals.     They  are  sometimes  straight  but  more  often 
somewhat  S-shaped  or  bent  on  themselves. 

[According  to  Eastwood  (working  for  the  English  Commission)  mammalian 
tubercle  bacilli  when  grown  on  serum  are  of  a  very  uniform  character  ; 
straight,  uniformly  stained  and  about  I/A  long  but  ranging  from  0'75-2'5/x. 
On  media  containing  glycerin  the  average  length  is  greater  :  in  the  same 
film,  short  (1/x)  bacilli,  longer  (2-4/A)  forms  and  very  long  (5-7  or  S/A)  forms 


FIG.  202. 


FIG.  203. 


FIG.  202.  —  Tubercle  bacilli  from  a  culture.  2  months  old,  on  glycerin-agar, 
of  a  typical  avian  virus.  In  addition  to  short  forms,  long  branching  forms  are 
found.  The  branching  frequently  emanates  from  a  darkly  stained  spherical 
point,  which  in  some  instances  is  of  a  greater  diameter  than  the  breadth  of  the 
bacillus.  Branching  is  much  commoner  with  the  avian  than  with  the  mam- 


malian bacillus.     Carbol-fuchsin. 


2150. 


(Eastwood.)     (See  footnote  p.  295.) 
FIG.  203.  —  Tubercle  bacilli  from  a  culture,  2  months  old,  on  glycerinated 
potato,  of  a  typical  avian  virus  (same  virus  as  fig.  202).     The  bacilli  show  a 
tendency  to  grow  in  long,  parallel  threads.     Carbol-fuchsin.      x  2150.     (East- 
wood.)    (See  footnote  p.  295.) 

may  all  be  encountered  and  on  these  media  the  bacilli  are  frequently  curved 
and  many  are  stained  irregularly  (beaded  and  globular  forms).  Branched 
forms  are  very  rarely  indeed  seen  in  cultures. 

[Avian  tubercle  bacilli  grown  on  glycerin  serum  are  generally  very  short 
(0'5-1/A)  and  rather  thick.  On  other  media  they  are  as  a  rule  longer  and 
more  irregular.  Among  these  irregular  forms  can  be  found  examples  of  all 
the  forms  assumed  by  the  mammalian  bacilli  ;  and  large  club-shaped  swellings 
are  not  uncommonly  seen  while 
branching  occurs  more  frequently 
than  with  the  mammalian  bacillus.  ] 

Koch  regarded  as  spores  the  un- 
stained intervals  which  are  some- 
times seen  in  the  bacilli.  There  is 
now  a  tendency  to  regard  the  deeply 
stained  granules  seen  at  the  ends  or 
in  the  length  of  some  bacilli  as 
spores  (Babes,  Ehrlich).  Gavina 
thinks  that  he  has  stained  terminal 
spores  in  bacilli  grown  in  presence  of 
antiseptics. 

In  cultures  extraordinarily  short 
bacilli    are    occasionally    seen. 
other  cases,  particularly  in  old  cul- 
tures, large,  branched  bacilli  endine 
in  a  club-shaped  swelling  are  foun 
(%.  204). 


These  giant  forms  afford 


FIG.  204. — Involution  forms  of  the  tubercle 
bacillus.     (After  Metchnikoff.) 


314  THE   TUBERCLE   BACILLUS' 

ground  for  grouping  the  tubercle  bacillus  with  the  streptothrices.  Coppen- 
Jones  [and  Eastwood]  have  described  ray  structures  with  club-shaped  ends  in 
tuberculous  lesions  exactly  similar  to  the  structures  seen  in  the  grains  of 
actinomycosis  (figs.  205  and  206). 

In  sputum  and  in  tuberculous  tissues  the  bacilli  are  found  singly  or  arranged 
in  groups  and  in  the  latter  case  may  lie  parallel  to  one  another.  Occasionally 
two  bacilli  are  seen  crossing  one  another  at  a  more  or  less  acute  angle  or 
arranged  like  a  V. 

[Bacilli  obtained  from  the  living  tissues  are  longer  and  not  so  uniform  in 
appearance  as  bacilli  cultivated  on  serum  (Eastwood). 

[Both  mammalian  and  avian  bacilli  when  growing  freely  in  the  tissues  of 
their  host  are  usually  shorter  and  more  uniformly  stained  than  those  which 
are  growing  under  adverse  conditions.] 

2.  Cultural  characteristics. 
A.  Conditions  of  growth. 

The  tubercle  bacillus  only  grows  in  artificial  culture  provided  that  the 
medium  contains  serum  (Koch),  glycerin  (Nocard  and  Roux),  yolk  of  egg 
(Dorset),  or  fragments  of  tissues  (Lumiere). 

It  is  an  aerobic  organism  and  only  grows  at  temperatures  above  30°  C.  •  In 
the  case  of  human  tubercle  bacilli  growth  ceases  at  41°  C.  and  in  the  case  of 
bovine  bacilli  at  44°-45°  C.  The  optimum  temperature  is  38°  C. 

Certain  precautions  must  be  observed  in  sowing  the  tubercle  bacillus.  For 
preference,  the  material  will  be  taken  from  a  lesion  in  the  guinea-pig  or  rabbit 
(bacilli  taken  directly  from  human  tissues  grow  badly  on  artificial  media), 
rubbed  up  in  a  sterile  mortar  and  portions  of  it  transferred  with  a  stout  wire  to 
tubes  of  coagulated  serum.  It  is  better  [when  sowing  cultures  of  the  human 
tubercle  bacillus]  to  use  serum  to  which  4  per  cent,  of  glycerin  has  been  added 
before  coagulation  or  blood  agar.  Never  sow  tuberculous  tissue  directly  on 
to  glycerin  agar :  the  cultures  are  more  than  likely  to  fail.  It  is  immaterial 
if  the  surface  of  the  medium  be  slightly  torn.  Sow  a  large  number  of  tubes 
as  many  of  them  will  remain  sterile  [and  others  are  likely  to  be  contaminated 
with  other  organisms].  Incubate  at  37°-38°  C.  Growth  only  becomes  visible 
to  the  naked  eye  after  an  interval  of  12  days  or  so  but  continues  to  increase 
for  about  4  weeks.  As  soon  as  colonies  appear  in  any  tube  cover  the  mouth 
with  an  india-rubber  cap  to  prevent  evaporation  and  the  consequent  drying 
up  of  the  medium.  [It  is  perhaps  even  better  to  seal  the  tube  with  paraffin 
or  sealing-wax.] 

When  a  growth  has  been  obtained,  sub-cultures  can  be  sown  on  various 
media ;  it  is  always  advisable  to  sow  a  good  deal  of  material  and  [until  a 
fair  amount  of  experience  has  been  acquired]  to  sow  several  tubes. 

[The  human  tubercle  bacillus  grows  more  luxuriantly  than  the  bovine 
tubercle  bacillus  in  artificial  culture  so  that  the  former  is  sometimes  described 
as  the  eugonic  and  the  latter  as  the  dysgonic  tubercle  bacillus.] 

Cultures  of  the  tubercle  bacillus  have  a  characteristic  but  rather  pleasant 
odour. 

B.  Characters  of  growth  on  various  media. 

1.  Coagulated  serum,  (a)  The  bacillus  of  the  human  type. — After  the 
culture  has  been  incubating  at  37°-38°  C.  for  12  days  or  so  a  number  of 
small,  white,  round,  scaly,  dry-looking  colonies  are  seen  scattered  over  the 
surface  of  the  medium.  On  further  incubation  the  colonies  become  raised 
but  retain  their  scaly  appearance,  and  the  margins  are  irregular  in  outline. 
Generally  speaking,  and  especially  when  recently  isolated,  the  colonies  do 


FIG.  205. — Lung  of  a  rabbit,  killed  63  days  after  inoculation,  partly  intra- 
venously and  partly  intra-peritoneally,  with  a  total  of  140  mg.  of  killed  culture 
of  bacilli  of  bovine  origin.  This  figure  is  a  representative  example  of  numerous 
lesions  found  in  the  lungs.  The  material  which  stains  strongly  with  eosin 
bears  a  curious  resemblance  to  actinomyces.  The  lesion  as  a  whole  is  abund- 
antly infiltrated  with  finely  granular  oxyphil  leucocytes.  As  carbol-fuchsin 
has  not  been  applied,  no  bacilli  are  stained.  Eosin  and  methylene  blue,  x  112. 
(Eastwood.)  (See  footnote  p.  295.) 


FIG  206  — From  the  same  lung  as  fig.  205,  the  specimen  having  been  stained 
with  carbol-fuchsin  before  counterstaining  with  eosin  and  methylene  blue. 
A  portion  of  one  of  the  actinomyces-like  nodules.  The  club  formation  may 
perhaps  be  attributable  to  dissolved  constituents  of  the  large  number  of  dead 
bacilli  inoculated.  Some  bacilli  not  yet  disintegrated  are  seen  in  the  lower 
part  of  the  field.  The  abundance  of  multinuclear  leucocytes,  which  are  shedding 
their  granules,  suggests  that  the  disintegration  of  the  dead  bacilli  is  due  to  the 
digestive  action  of  these  cells.  Carbol-fuchsin,  eosin  and  methylene  blue. 
x  865.  (Eastwood.)  (See  footnote  p.  295.) 


316 


THE  TUBERCLE   BACILLUS 


not  become  confluent.     After  sub-cultivating  three  or  four  times,  however, 
they  may  coalesce  to  form  a  dried  wrinkled  layer. 

[(/?)  The  bovine  type. — On  pure  serum  the 
growth  of  the  bovine  tubercle  bacillus  pre- 
sents no  marked  differences  from  that  of 
the  human  tubercle  bacillus.  ] 

(y)  The  avian  type. — The  bacillus  of  avian 
origin  yields  a  more  abundant  growth  on 
serum  than  the  human  bacillus.  The  culture 
is  thick  and  generally  has  a  moist,  greasy 
lustre. 

2.  Glycerin  agar. — Except  for  primary 
cultures,  this  is  the  best  medium  upon 
which  to  grow  the  tubercle  bacillus.  A  little 
glucose  may  with  advantage  be  added  to  the 
medium  (p.  44). 

(a)  The  human  type. — Growth  begins  as 
on  serum  but  the  colonies  are  both  larger 
and  more  numerous.  They  rapidly  become 
confluent  and  form  a  thick,  whitish,  dry, 
rough,  scaly,  mammilated  layer  even  in 
recently  isolated  specimens  and  after  being 
sub-cultivated  a  few  times  on  glycerin-agar 
the  growth  becomes  very  abundant,  moist, 
greasy  and  folded.  When  old  the  growth 
has  a  reddish  tint. 

[(/3)  The  bovine  type. — The  bovine  tu- 
bercle bacillus  grows  much  less  luxuriantly 
on  media  containing  glycerin — such  as 
glycerin-serum,  glycerin-agar,  or  glycerin- 
potato — than  the  human  tubercle  bacillus 
(English  Commission).] 

(y)  The  avian  type.— Some  authors  have 
contrasted  the  growth  of  the  avian  bacillus 
on  glycerin-agar  with  that  of  the  human 
bacillus.  The  latter,  they  say,  gives  rise  to 
a  dried,  wrinkled  layer,  while  the  former 
produces  a  moist,  fatty  growth.  But  as  has 
been  seen,  the  human  type  frequently  gives 
a  copious  growth  of  a  moist,  fatty  appear- 
ance :  and  it  is  equally  true  that  the  avian 
type  occasionally  produces  a  dry,  scaly 
growth  (Nocard,  Grancher,  Fischel). 

["  The  most  characteristic  point  of  difference  between  the  mammalian  and 
avian  tubercle  bacilli  is  that  the  cultures  of  avian  bacilli  are  moist  and  easily 
emulsified,  while  on  most  media  the  mammalian  cultures  are  dry  and  can 
only  be  broken  up  with  difficulty  "  (F.  Griffith,  for  the  English  Commission).] 
3.  Egg  medium. — A  useful  medium  for  the  growth  of  the  tubercle  bacillus 
consists  of  the  white  and  yolk  of  eggs  coagulated  and  sterilized  by  discon- 
tinuous heating  at  72°-74°  C.  (Dorset,  Capaldi,  A.  S.  Griffith). 

Bezan9on  and  Griffon  mix  one  part  of  uncooked  yolk  of  egg  with  two  parts 

[^Fig.  207  and  also  figs.  208,  209,  210,  and  211  are  from  the  Final  Report  of  the 
Royal  Commission  on  Tuberculosis  (Human  and  Bovine) — Part  II.  Appendix,  Vol.  I. ; 
Dr.  A.  Stanley  Griffith — by  permission  of  the  Controller  of  H.M.  Stationery  Office.] 


(a)  (ft) 

4th  Generation  4th  Generation 

51  days  old.  3  weeks  old. 

FIG.  207. — Tubercle  bacilli  of  human 
origin  cultivated  on  glycerin-agar.  (a) 
A  culture  from  the  tuberculous  mesenteric 
glands  of  a  child  aged  8i  years,  who  died 
from  multiple  stricture  of  the  gut  (due 
to  tuberculous  ulceration).  The  culture 
grew  moderately  well  on  artificial  media, 
and  resembled  the  more  easy-growing  cul- 
tures of  bovine  origin ';  it  had  high  viru- 
lence for  the  calf,  rabbit  and  guinea-pig. 
(6)  A  culture  from  a  mesenteric  gland  of 
a  case  of  general  tuberculosis  originating 
in  the  alimentary  tract  in  a  child  aged 
10  months.  The  strain  grew  luxuriantly 
on  media  containing  glycerin  and  was 
only  slightly  virulent  for  the  rabbit  (A.  S. 
Griffith).i 


CULTURAL  CHARACTERISTICS 


317 


of  6  per  cent,  glycerin-agar  melted  in  a  water  bath  and  kept  at  50°  C.  The 
ingredients  are  mixed  as  thoroughly  as  possible  and  the  tubes  allowed  to  set 
in  a  sloped  position.  From  human  lesions,  moist,  greasy-looking  colonies 
can  be  obtained  in  a  week  on  this  medium. 

Phisalix  prepares  a  medium  by  mixing  yolk  of  egg  with  a  potato  mash 
containing  a  little  glycerin.  The  medium  is  sterilized  in  the  autoclave. 
Lubenau  gives  a  method  which  has  already  been  described  (p.  54). 

According  to  Park,  yolk  of  egg  media  are  particularly  useful  for  the  isola- 
tion of  tubercle  bacilli 'from  tuberculous  material  and  for  the  differentiation 

of  the  human  from  the   bovine    type.      

On  media  made  with  yolk  of  egg  but 
containing  no  glycerin  bacilli  of  the 
bovine  type  grow  easily  :  and  on  the 
same  media  but  containing  glycerin 
bacilli  of  the  human  type  grow  poorly 
at  first  while  the  bovine  type  does  not 
grow  at  all. 

[According  to  A.  S.  Griffith  (English 
Commission)  the  egg  medium  is  in- 
valuable for  obtaining  the  tubercle 
bacillus  in  pure  culture  from  tissues  or 
other  material.  It  is  of  great  value  also 
for  sub-cultures ;  on  it  the  tubercle 
bacillus  retains  its  vitality  for  a  longer 
period  than  on  any  other  medium,  and 
sub-cultures  can  often  be  obtained  on 
this  medium  from  old  cultures  which 
fail  to  grow  when  sown  on  other  media. 
He  gives  the  following  method  of  pre- 
paration of  the  medium — "  The  yolk  and 
the  white  of  fresh  eggs  are  thoroughly 
mixed  by  shaking  in  a  flask ;  salt  solution 
is  added  in  the  proportion  of  one  to  three 
of  egg ;  the  mixture  is  filtered  through 
muslin,  distributed  into  tubes,  sloped  and 
coagulated  in  a  serum  inspissator  at 
80°  C."] 

4.  Blood-agar. — -Bezancon  and  Griffon 
recommend  the  addition  of  rabbit-blood 
to  agar  for  starting  cultures  from  human 
or  animal  tissues.     Growth  appears  early 
and    soon   becomes   very   copious,    the 
colonies    absorb    the   haemoglobin    and 
become  chocolate-coloured. 

5.  Tochtermann's  agar. — Dissolve  10  grams  of  peptone,  5  grams  of  sodium 
chloride,  5  grams  of  glucose  and  20  grams  of  agar  in  a  litre  of  water.     Add 
half-a-litre  of  calf  serum,  mix,  boil  for  15  or  30  minutes,  filter  in  the  warm, 
distribute  into  tubes  and  sterilize  at  100°  C.  for  50  minutes. 

6.  Hesse's  agar. — For  obtaining  cultures  of  the  tubercle  bacillus  from  human 
sputum  Hesse  recommends  sowing  the  material  on  the  surface  of  a  special 
agar  prepared  as  follows  : 

Dissolve  5  grams  of  salt,  30  grams  of  glycerin,  and  20  grams  of  agar  in  a 
litre  of  water.  Add  5  c.c.  of  a  normal  solution  of  carbonate  of  sodium  and  5 
grams  of  Heyden's  albumose  (Nahrstoff  Heyden)  dissolved  in  50  c.c.  of  water. 


(a)  (6) 

FIG.  208.— Primary  cultures  of  tubercle  bacilli 
on  the  egg  medium .  (a)  Bovine  tubercle  bacilli 
of  human  origin  obtained  direct  from  sputum 
bv  means  of  antiformin  :  (6)  Human  tubercle 
bacilli  (A.  S.  Griffith).  (See  footnote  p.  316.) 


318  THE   TUBERCLE   BACILLUS 

Boil  for  15  minutes.     Filter  in  the  warm.     Sterilize  at  100°  C.  and  pour 
into  Petri  dishes. 

7.  Fragments  of  tissue  as  culture  media. — A.  and  L.  Lumiere  obtain  very 
copious  growths  commencing  in  about  36  hours  on  fragments  of  liver  or 
spleen. 

Wash  the  liver  and  spleen  of  an  adult  bovine  animal  or  calf  in  distilled  water, 
heat  in  the  autoclave  for  three-quarters  of  an  hour  to  shrink  them  and  then  cut 
into  rectangular  pieces ;  after  soaking  in  a  6  per  cent,  aqueous  solution  of  glycerin 
for  an  hour  the  pieces  are  placed  in  potato  tubes  and  sterilized  in  the  autoclave  for 
15  minutes.  It  is  best  to  sow  from  a  potato  culture. 

Gioelli  uses  pieces  of  human  placenta  immersed  in  broth,  or,  better,  placenta- 
broth  containing  O5  per  cent,  of  sodium  chloride  and  6  per  cent,  glycerin 
in  place  of  liver. 

8.  Bile. — Calmette  recommends  pieces  of  potato  soaked  in  a  mixture  of 
95  parts  fresh  bile  and  5  parts  sterilized  glycerin.     The  bile-glycerin  mixture 
ought  to  be  kept  for  3  weeks  at  the  temperature  of  the  laboratory  before  being 
used.     [On  an  ox-bile-glycerin-potato  medium  tubercle  bacilli  of  the  bovine 
type  grow  more  rapidly  and  more  luxuriantly  than  on  the  usual  media  while 
bacilli  of  the  human  type  grow  with  difficulty  on  this  medium  and  the  avian 
type  not  at  all.     On  the  other  hand  bacilli  of  the  human  type  will  grow 
rapidly  on  an  human-bile  medium  as  will  bacilli  of  the  avian  type  on  a  fowl- 
bile  medium  ;  the  cultivations  of  these  two  types  on  these  media  respectively 
are  similar  in  appearance  to  cultivations  of  the  bovine  type  on  ox-bile 
(Calmette)]. 

9.  Glycerin-broth. — Glycerin-broth,  or  better,  glucose-glycerin-broth  is  a 
very  useful  medium  for  the  growth  of  the  tubercle  bacillus.     To  sow  a 
broth  culture  of  the  tubercle  bacillus,  raise  as  large  a  piece  of  growth  as 
possible  from  the  surface  of  an  agar  tube  or  other  solid  medium  (it  is  even 
better  to  lift  the  film  of  growth  from  the  water  of  condensation)  with  a  fairly 
large  platinum  loop  and  float  it  very  carefully  on  to  the  surface  of  the  broth. 
It  is  advisable  to  transfer  three  or  four  loopsful  if  a  large  flask  is  to  be  sown. 

Growth  generally  takes  place  in  the  form  of  a  pellicle.  After  incubating 
for  about  a  fortnight  a  whitish  area  appears  around  the  piece  of  growth 
which  was  sown,  this  gradually  extends  and  ultimately  forms  a  thin  delicate 
film  covering  the  whole  of  the  surface  of  the  medium.  The  film  is  at  first 
dry  and  fragile  but  becomes  thicker  in  course  of  time  :  sometimes  it  remains 
dry  and  scaly  and  sometimes  becomes  greasy,  moist  and  wrinkled.  Not 
infrequently  the  film  creeps  up  the  sides  of  the  vessel,  sometimes  to  a  height 
of  1  cm.  Rarely  no  film  at  all  is  formed  and  in  this  case  the  growth  consists 
of  a  flocculent  deposit.  Whatever  the  form  of  growth  the  broth  remains 
quite  clear. 

[To  obtain  a  successful  growth  on  glycerin-broth  requires  considerable 
attention  to  details.  The  material  used  for  sowing  the  medium  must  be 
young  and  actively  growing — perhaps  the  film  growing  on  the  water  at 
the  bottom  of  a  glycerin-potato  culture  gives  the  best  results.  If  the  culture 
be  sown  at  the  right  moment  the  growth  will,  in  the  case  of  the  human  type, 
spread  and  cover  the  surface  of  the  glycerin-broth  in  a  Roux's  bottle  laid 
on  its  flat  side  in  a  fortnight.  At  other  times  no  change  whatever  is  visible 
in  the  material  sown  for  weeks,  then  little  white  nodules  appear  which  are 
the  precursors  of  a  growth  which  once  it  starts  to  spread  covers  the  surface 
very  rapidly.  The  flask  must  be  carefully  sealed.  ] 

The  tubercle  bacillus  can  also  be  grown  on  ordinary  broth  containing  no  glycerin 
(Gioelli  [and  others]). 

Pour  a  layer  of  vaseline  oil  about  1  mm.  deep  on  the  surface  of  the  broth.     Sterilize 


and 


CULTURAL  CHARACTERISTICS 


319 


and  sow  the  medium  from  an  agar  culture  or  with  pieces  of  tuberculous  tissue  which 
have  been  carefully  crushed.  The  material  used  for  sowing  should  float  on  the  sur- 
face of  the  vaseline  or  between  the  vaseline  and  the  broth.  Should  it  fall  to  the 
bottom  it  is  only  necessary  to  shake  the  flask  carefully  and  the  drops  of  oil  will 
float  the  material  to  the  surface  again.  To  make  microscopical  preparations  blot 
up  the  oil  from  the  slide  with  blotting  paper. 


7th  Generation 

5  weeks  old. 

FIG.  209. — A  culture  of  the  human  tubercle  bacillus  on  glycerin  broth.  The 
culture  was  isolated  from  the  lung  of  a  man  aged  33,  who  died  of  phthisis ;  it 
grew  luxuriantly  on  all  media  containing  glycerin,  was  virulent  for  chimpanzees, 
monkeys  and  guinea-pigs  but  had  only  slight  virulence  for  calves,  rabbits  and 
horses  (A.  S.  Griffith).  (See  footnote  p.  316.) 

10.  Glycerin-fish-broth. — This  medium  has  been  recommended  by  Martin. 
The  cultural  characteristics  are  the  same  as  on  ordinary  glycerin-broth. 

Mince  the  flesh  of  an  herring  and  add  to  it  one  and  half  times  its  weight 
of  water.  Heat  slowly  and  keep  it  boiling  for  three-quarters  of  an  hour. 
Filter  through  Chardin  paper  several  times  while  warm  and  when  clear  add 
6  per  cent,  of  glycerin.  Neutralize  if  necessary.  Distribute  in  tubes  and 
sterilize  in  the  autoclave. 


320 


THE   TUBERCLE   BACILLUS 


11.  Potato. — The  tubercle  bacillus  will  grow  on  ordinary  potato  but  to 
obtain  the  best  results  it  is  necessary  to  add  glycerin  (Nocard). 

Cut  the  potatoes  into  suitably-shaped  pieces  and  leave  them  to  soak  in  a 
large  quantity  of  a  15  per  cent,  solution  of  glycerin  in  water  for  two  days  in 
the  ice  chest,  then  transfer  them  to  a  number  of  Roux's  tubes  and  sterilize 
in  the  ordinary  way. 

On  this  medium  growth  appears  about  the  twelfth  day  generally  taking 
the  form  of  a  thick,  folded,  soft  layer,  very  occasionally  it  is  dry  and  wrinkled. 
The  growth  often  extends  in  the  form  of  a  film  over  the  liquid  which  has 
drained  away  into  the  lower  part  of  the  tube.  This  film  will  be  found  very 
useful  for  sowing  liquid  cultures. 


(a) 

llth  Generation 
70  days  old. 


12th  Generation 
30  days  old. 


FIG.  210. — Cultures  of  tubercle  bacilli  of  human  origin  on  glycerin-potato, 
(a)  A  bovine  tubercle  bacillus  isolated  from  the  meninges  of  a  child  aged  2  years 
who  died  of  tuberculous  meningitis.  The  culture  proved  highly  virulent  for 
the  calf,  rabbit,  guinea-pig  and  cat.  (6)  An  human  tubercle  bacillus  isolated 
from  the  sputum  of  a  youth  aged  16  years  suffering  from  pulmonary  tuber- 
culosis. The  culture  grew  luxuriantly  on  artificial  media  and  was  slightly 
virulent  for  rabbits  (A.  S.  Griffith).  (See  footnote  p.  316.) 

Jurewitch  recommends  a  potato  broth.     Leave  a  potato  mash  to  macerat 
in  its  own  weight  of  water  for  a  day,  then  filter  through  muslin  and  to  the 
filtrate  add  an  equal  volume  of  meat  extract,  0*5  per  cent,  peptone  (Chapo- 
teaut),  0'5  per  cent,  salt  and  3  per  cent,  glycerin  :    make  distinctly  alkaline 
and  complete  the  preparation  as  in  making  ordinary  broth  (p.  30). 

C.  Differentiation  of  the  various  types  of  tubercle  bacilli  by  cultural  methods. 

[A.  S.  Griffith  (for  the  English  Commission)  investigating  the  cultural 
characters  of  mammalian  bacilli,  proceeds  as  follows.  From  the  primary 
culture  on  egg  the  bacillus  is  transferred  to  pure  serum  until  it  is  growing 
vigorously  :  it  is  then  tested  on  the  differential  media.  On  these  media  human 
tubercle  bacilli  produce  luxuriant  growths  while  bovine  tubercle  bacilli  grow 
much  less  luxuriantly.  It  is  possible  thus  to  determine  the  type  of  bacillus  by 
cultural  characteristics  alone.  The  differential  media  are  serum,  agar,  potato 
and  broth,  all  containing  glycerin.  The  serum,  agar  and  broth  contain  5  per 


cei 


CULTURAL  CHARACTERISTICS 


321 


cent,  glycerin  and  are  prepared  in  the  customary  way.  "  The  potato  is  cut  into 
slices  put  in  water  and  soaked  for  24  hours  in  a  5  per  cent,  glycerin  blue- 
litmus  solution,  which  is  poured  off  before  the  first  sterilization  ;  at  the  bottom 
of  each  tube  is  some  absorbent  cotton-wool  soaked  with  glycerin  solution 
which  helps  to  keep  the  potato  moist." 


5th  Generation 
over  3  months  old. 


6th  Generation 
55  days  old. 


FIG.  211. — Cultures  of  tubercle  bacilli  of  human  origin  on  glycerin-gelatin, 
(a)  A  bovine  tubercle  bacillus  of  intermediate  virulence  for  calves  and  rabbits  ; 
(6)  an  human  tubercle  bacillus  slightly  virulent  for  rabbits  (A.  S.  Griffith). 
(See  footnote  p.  316.) 

[F.  Griffith  states  that  "it  is  possible  to  differentiate  between  the  avian 
and  the  mammalian  types  of  tubercle  bacilli  from  cultural  characters  alone, 
but  it  is  necessary  in  order  to  avoid  error,  that  a  sufficient  variety  of  media 
should  be  used.  In  primary  cultures  on  serum  or  serum  agar  the  transparent 
colonies  of  avian  tubercle  bacilli  are  easily  recognized.  On  glycerin-agar 


322  THE  TUBERCLE   BACILLUS 

and  glycerin-potato  the  avian  bacillus  frequently  forms  a  wrinkled  or  warty 
growth  resembling  a  culture  of  human  tubercle  bacilli ;  but  the  characteristic 
difference  is  evident  when  the  growth  is  touched  with  the  spatula."] 


SECTION   III.— BIOLOGICAL   PROPERTIES. 
1.  Viability  and  virulence. 

In  determining  the  viability  of  the  tubercle  bacillus,  animal  inoculation, 
not  artificial  cultivation,  must  be  the  test  [because  sub-cultures  often  fail — 
though  the  culture  used  for  sowing  them  may  be  alive — especially  if  the 
operator  has  not  had  considerable  experience  in  dealing  with  cultures  of  the 
tubercle  bacillus]. 

In  cultures,  the  tubercle  bacillus  is  only  slightly  resistant  to  adverse  con- 
ditions and  this  is  one  of  the  arguments  in  favour  of  the  view  that  it  does 
not  form  spores.  Exposure  to  70°  or  75°  C.  for  10  minutes  kills  cultures  of 
the  bacillus  in  liquid  media. 

[F.  Griffith  (for  the  English  Commission)  made  numerous  tests  on  bacilli 
grown  on  various  media  and  concludes  that  "  cultures  "  (of  mammalian  or 
avian  tubercle  bacilli)  "  will  maintain  their  vitality  for  long  periods  whether 
kept  in  the  incubator  or  at  room  temperature."  Avian  bacilli  in  one  culture- 
tube  were  found  alive  after  1067  days,  and  bovine  bacilli  similarly  after 
990  days  :  "  but  no  distinction  could  be  drawn  between  the  types  of  bacilli 
tested."] 

Agar  cultures  [are  said  to]  lose  their  virulence  after  a  few  months  [but 
F.  Griffith  found  that  "  little  if  any  attenuation  "  (of  the  bovine  bacillus) 
"  was  caused  by  residence  on  artificial  media  "  (serum  and  glycerin-serum) 
for  periods  up  to  1487  days.  Human  tubercle  bacilli  tested  after  from  2-3 
years'  artificial  cultivation  (on  serum)  showed  no  appreciable  diminution  in 
virulence  (A.  S.  Griffith).] 

[Calmette  and  Guerin  state  that  cultures  on  glycerin-bile-potato  (p.  318) 
are  at  first  increased  in  virulence  but  that  repeated  sub -cultivation  on  that 
medium  diminishes  the  virulence  of  the  bacilli  for  certain  animal  species.] 

In  moist  sputum  the  bacillus  is  not  destroyed  at  75°  C.  but  is  killed  in 
5  minutes  at  a  temperature  of  100°  C. 

Cultures  of  the  tubercle  bacillus,  or  tubercle  bacilli  in  sputum,  retain  their 
vitality  for  a  long  period  when  dried  at  the  ordinary  temperature  of  the  air. 
Under  these  conditions,  the  bacilli  may  retain  their  virulence  for  several 
months  (Galtier)  ;  they  are  not  destroyed  by  exposure  to  a  dry  temperature 
of  100°  C.  for  2  or  3  hours  and  are  capable  of  resisting  a  temperature  of  70°  C. 
for  more  than  7  hours  (Welch,  Grancher,  Ledoux-Lebard), 

The  combined  action  of  desiccation  and  sunlight  [is  said  to], attenuate  the 
virulence  of  the  bacillus  (Candler,  Koch,  Migneco  and  Kansome). 

Zilgen  mixed  some  dust  with  dried  tuberculous  sputum  and  exposed  the 
mixture  to  the  action  of  sunlight :  under  these  conditions,  the  tubercle 
bacillus  retained  its  virulence  for  about  140  days.  According  to  de  Thoma 
the  virulence  of  the  bacilli  in  sputum  left  in  the  patient's  room  disappears 
after  two  months  and  a  half  but  is  retained  indefinitely  when  the  sputum  is 
kept  in  the  dark  under  the  same  conditions. 

The  virulence  of  tubercle  bacilli  in  sputum  exposed  to  the  alternate  action 
of  moisture  and  desiccation  is  retained  for  several  months  (Malassez  and 
Vignal). 

The  tubercle  bacillus  appears  to  maintain  its  vitality  in  water  for  a  long 
time  :  it  has  been  recovered  from  sterile  water  after  70  days  (Chantemesse 


BIOLOGICAL  PROPERTIES  323 


and  Widal)  and  after  exposure  to  running  water  for  150  days  (Cadeac  and 
Malet). 

Putrefaction  has  little  effect  on  the  tubercle  bacillus.  Tuberculous  tissues 
left  to  decompose  in  water  for  20  and  40  days  did  not  lose  their  virulence 
(Gal tier).  Tuberculous  lungs  buried  for  167  days  have  been  found  to  be 
virulent  (Cadeac  and  Malet).  Schottelius  found  that  the  bacillus  was  virulent 
in  tissues  which  had  been  buried  for  2  years.  Gsertner  made  the  same  observa- 
tion after  burying  the  tuberculous  tissues  for  a  winter. 

Action  of  antiseptics. — In  cultures,  the  tubercle  bacillus  is  somewhat 
sensitive  to  the  action  of  antiseptics.  According  to  Yersin  it  is  killed  in 
30  seconds  in  a  5  per  cent,  solution  of  phenol ;  in  5  minutes  in  absolute 
alcohol,  and  in  1  per  cent,  iodoformed  ether  ;  in  10  minutes  in  a  1  in  1000 
solution  of  perchloride  of  mercury,  and  after  several  hours  in  0'3  per  cent, 
thymol  or  0'25  per  cent,  salicylic  acid.  It  resists  the  action  of  4  per  cent, 
boric  acid  for  more  than  12  hours. 

According  to  Koch,  the  following  substances  readily  hinder  the  growth  of 
the  bacillus  in  cultures,  viz.  : — the  essential  oils,  naphthol,  fuchsin,  methy- 
lene  blue,  gentian-violet,  and  especially  the  cyanides  of  gold  and  silver. 
Cyanide  of  gold  in  the  proportion  of  1  part  in  2  millions  stops  the  growth  of 
the  bacillus. 

But  in  tuberculous  fluids  and  tissues  the  resistance  of  the  bacillus  is  much 
greater  :  thus  1  in  500  salicylic  acid,  1  in  1000  bromine,  creosote,  quinine, 
1  in  1000  perchloride  of  mercury,  and  formalin  vapour  have  no  effect  on  the 
bacillus.  Six  per  cent,  carbolic  acid  has  a  doubtful  influence,  a  1  in  4000 
solution  of  hydrofluoric  acid  (a  very  caustic  liquid)  has  hardly  any  action 
(H.  Martin).  The  numerous  experiments  of  Vallin,  Mairet,  Cavalier,  Coze 
and  Siamon  and  others  have  given  contradictory  results. 


2.  Toxins. 
A.  Toxic  properties  of  dead  tubercle  bacilli. 

Koch  and  many  other  observers  have  found  that  agar  cultures  sterilized 
by  heat  at  100°  C.  are  injurious  to  animals,  and  that  in  sufficiently  large 
doses  they  lead  to  suppuration,  cachexia  and  death  in  guinea-pigs.  Dead 
tubercle  bacilli  inoculated  into  the  blood  or  peritoneal  cavity  [of  the  guinea- 
pig]  lead  to  the  formation  of  true  tubercles  in  which  the  presence  of  the 
dead  bacilli  can  be  demonstrated,  but  these  lesions  do  not  become  generalized 
nor  are  they  capable  of  being  passed  on  to  another  animal.  When  only 
a  small  dose  of  dead  bacilli  is  inoculated  the  lesions  disappear  spontaneously 
after  a  few  months  and  the  animal  recovers  (Cautacuzene).  The  more 
virulent  the  living  bacilli  the  more  toxic  the  dead  bacilli. 

[F.  Griffith  (English  Commission)  observing  that  the  intra- venous  inocu- 
lation of  living  mammalian  tubercle  bacilli  in  doses  of  10-50  mg.  caused 
death  in  about  50  per  cent,  of  fowls,  inoculated  3  fowls  each  with  10  mg.  of 
bovine  tubercle  bacilli  killed  by  exposure  to  steam  at  100°  C.  and  found  in 
the  lungs  of  two  of  them  after  38  days  numerous  minute  caseating  tubercles. 
He  concluded  that  the  fatal  infections  were  due  to  the  direct  toxic  action  of 
the  bacilli  and  were  not  true  tuberculosis. 

[Similar  results  were  obtained  in  fowls  inoculated  with  dead  human  and 
avian  tubercle  bacilli ;  the  latter  were  not  more  toxic  for  the  fowl  than  the 
mammalian  types.] 

Hammerschlag  treated  dried  tubercle  bacilli  with  alcohol  and  ether.  He 
obtained  a  product  which  was  toxic  to  guinea-pigs  and  rabbits.  The  inocu- 
lated animals  had  convulsions  and  died. 


324  THE  TUBERCLE   BACILLUS 

Auclair  extracted  tubercle  bacilli  with  ether  and  with  chloroform.  On 
inoculation  into  the  trachea  of  a  guinea-pig,  these  extracts  produced  lesions 
of  tuberculous  pneumonia. 

Borrel  also  extracted  a  toxic  substance  from  tubercle  bacilli  with  xylol. 

8.  Koch's  old  tuberculin. 

The  tuberculin  prepared  by  Koch  in  1890  by  a  method  at  first  kept  secret 
is  prepared  in  the  following  manner  at  the  Pasteur  Institute  in  Paris. 

A  culture  of  the  tubercle  bacillus  on  glycerin-broth  is  grown  in  a  flask. 
(Bacilli  of  mammalian  and  avian  origin  yield  the  same  tuberculin.)  [Certain 
experiments  recorded  by  the  English  Commission  however  show  that  tuber- 
culin of  avian  origin  cannot  be  relied  on  to  produce  a  reaction  in  animals 
suffering  from  mammalian  tuberculosis.  ]  The  growth  must  be  on  the  surface 
of  the  broth.  The  film  appears  after  incubating  for  15-20  days  at  38°  C. 
and  is  complete  about  the  40th  day. 

The  whole  is  sterilized  at  110°  C.  for  15  minutes,  then  evaporated  on  a 
water  bath  to  one-tenth  its  volume,  and  filtered  through  filter  paper.  The 
filtrate  constitutes  crude  tuberculin. 

Tuberculin  is  a  brownish  syrupy  liquid  with  a  faint,  pleasant,  characteristic 
smell.  It  has  no  definite  composition  but  is  a  simple  extract  prepared  from  sterilized 
cultures  on  glycerin-broth,  and  contains  in  addition  to  the  products  secreted  by  the 
bacilli  the  substances  originally  present  in  the  broth.  The  active  principle  has  not 
yet  been  extracted.  Tuberculin  is  very  resistant  to  heat  but  is  destroyed  at  150°  C. 

Attempts  have  been  made  to  purify  the  crude  product : 

(a)  On  the  addition  of  20  volumes  of  strong  alcohol  a  brown  precipitate  is  thrown 
down  which  contains  the  active  principle  and  a  number  of  other  extraneous  sub- 
stances. Tannin,  picric  acid,  metallic  salts,  ferrocyanide  of  potassium  and  acetic 
acid  also  form  an  albuminoid  precipitate  which  carries  down  the  active  principle. 
Koch,  Hunter  and  Klebs  have  failed  in  their  attempts  to  purify  this  precipitate. 

(6)  Koch  precipitated  crude  tuberculin  with  three  volumes  of  66  per  cent,  alcohol 
and  obtained  a  flocculent  precipitate  which  on  drying  formed  a  white  powder.  This 
constitutes  purified  tuberculin  :  it  contains  numerous  extraneous  substances  but 
is  a  very  toxic  product  and  kills  guinea-pigs  when  inoculated  in  doses  of  1  mg.  This 
is  a  very  expensive  method  of  preparation,  as  nine-tenths  of  the  tuberculin  remain 
in  solution  and  are  lost.  Calmette  precipitates  crude  tuberculin  with  95  per  cent, 
alcohol. 

'Except  for  Calmette's  ophthalmo-reaction  no  advantage  is  to  be  gained 
by  using  purified  tuberculin — it  has  the  same  properties  as  crude  tuberculin. 

1.  The  effect  of  Koch's  old  tuberculin  on  man  and  animals. 

1.  Healthy  (non-tuberculous)  subjects. — Crude  tuberculin  inoculated  into 
healthy  animals  in  small  doses  has  no  untoward  effect  except,  possibly,  a 
very  slight  rise  of  temperature.  Guinea-pigs  can  be  inoculated  with  2  c.c. 
of  tuberculin  without  harm.  Rabbits  stand  an  injection  of  5  c.c.  of  crude 
tuberculin  very  well,  there  is  a  slight  rise  of  temperature  and  a  transitory 
loss  of  weight  but  the  animal  quickly  recovers.  Cattle  and  dogs  do  not 
react  to  doses  of  10  c.c. 

In  man  an  injection  of  0'25  c.c.  into  a  healthy  adult  leads  to  somewhat 
severe  symptoms :  rigors,  diarrhoea  and  vomiting  with  a  rise  of  temperature 
to  perhaps  39°  C.  (Koch).  As  small  a  dose  as  O'Ol  c.c.  may  produce  a  slight 
rise  of  temperature.  Man  is  therefore  about  1000  to  1500  times  as  sensitive 
to  tuberculin  as  the  guinea-pig. 

The  toxicity  of  tuberculin  can  be  considerably  diminished  by  adding  to  it  a  calcu- 
lated amount  of  anti-tuberculous  serum  (vide  infra),  the  toxicity  of  the  mixture  is 
then  due  to  toxones.  Tuberculin  neutralized  in  this  way  gives  no  better  results  in 


the 


KOCH'S   OLD  TUBERCULIN  325 


the  treatment  of  tuberculosis  than  tuberculin  or  anti-tuberculous  serum  given  alone. 
It  [is  said  to]  assist  the  production  of  the  disease  experimentally  (Arloing  and 
Descos). 

2.  In  persons  infected  with  tuberculosis  and  in  tuberculous  animals,  the 
inoculation  of  small  doses  of  tuberculin  gives  rise  to  a  marked  reaction  and 
ere  symptoms  which  may  terminate  fatally. 

A  dose  of  O5  c.c.  of  tuberculin  rapidly  kills  a  guinea-pig  infected  with 
tuberculosis  5  or  6  weeks  before  ;  there  is  a  sudden  rise  of  temperature  followed 
by  a  gradual  fall  and  the  animal  dies  in  a  state  of  coma.  Post  mortem  there 
is  intense  congestion  around  the  tuberculous  foci  and  the  internal  organs  are 
red,  congested  and  ecchymosed. 

In  tuberculous  cattle  the  inoculation  of  quantities  of  0* 30-0' 40  c.c.  causes 
a  rise  of  temperature  about  6  hours  afterwards  from  38°-39°  C.  (the  normal 
bovine  temperature)  to  40°-41°  C.  The  animal  recovers  its  normal  condition 
in  a  few  days.  Large  doses  of  tuberculin  are  liable  to  kill  the  animal. 

Persons  suffering  from  tuberculosis  react  very  sharply  indeed  to  the 
inoculation  of  tuberculin  :  O25  c.c.  invariably  leads  to  a  fatal  result. 

The  so-called  curative  doses  employed  by  Koch  were  G'003-0'004  c.c.  Following 
the  injection  the  patient  had  rigors,  and  a  rise  of  temperature  to  41°  C.  The  inocu- 
lation was  frequently  followed  by  coughing,  nausea,  vomiting,  jaundice,  etc.  Around 
cutaneous  tuberculous  lesions  there  was  an  intense  inflammatory  reaction.  Accord- 
ing to  Koch  these  symptoms  ought  to  last  12—15  hours  and  then  give  way  to  a  pro- 
gressive improvement  of  the  pre-existing  lesions.  Nothing  would  be  gained  here 
by  recalling  the  disasters  which  followed  the  use  of  tuberculin.  The  treatment  of 
tuberculosis  with  tuberculin  has  recently  been  revived  especially  in  Germany  as 
the  result  of  the  work  of  Denys,  Sahli,  and  Beraneck.  The  doses  used  are  much 
smaller  than  those  used  by  Koch.  The  results  obtained  by  this  method  of  treat- 
ment have  not  shown  it  to  be  of  any  great  value. 

Intra-cerebral  inoculation. — A  guinea-pig  weighing  500  grams  which  will 
stand  the  inoculation  of  1  c.c.  sub-cutaneously  dies  when  inoculated  in  the 
brain  with  3-4  mg.  of  the  same  tuberculin  (von  Lingelsheim,  Borrel). 

A  guinea-pig  inoculated  12  days  previously  with  tuberculous  material 
succumbs  to  the  intra-cerebral  inoculation  of  O'l  mg.  of  tuberculin.  The 
inoculation  of  0*001  mg.  of  tuberculin  into  the  brain  of  a  guinea-pig  which 
has  been  infected  with  tuberculosis  6  weeks  previously  produces  symptoms 
of  hiccough,  convulsions,  muscular  twitchings,  etc.  and  the  animal  very  soon 
dies.  These  facts  afford  an  explanation  of  the  symptoms  of  tuberculous 
meningitis — the  only  form  of  tuberculosis  in  which  the  action  of  the  poison  on 
the  nerve  cells  can  be  demonstrated  (Borrel). 

The  toxins  of  tetanus,  plague,  etc.,  are  no  more  toxic  in  the  brain  of  tuberculous 
guinea-pigs  than  in  the  brain  of  healthy  animals.  Mallein  alone  acts  like  tuberculin  : 
unconcentrated  mallein  which  is  harmless  to  tuberculous  guinea-pigs  when  inoculated 
sub-cutaneously  in  doses  of  3-4  c.c.  leads  to  a  fatal  result  when  inoculated  intra- 
cerebrally  in  doses  of  O'Ol  or  O'OOl  c.c.  (Borrel). 

2.  Koch's  old  tuberculin  in  the  diagnosis  of  tuberculosis. 

A.  In  Cattle. — Nocard  showed  that  tuberculin  is  a  valuable  reagent  in  the 
diagnosis  of  tuberculosis  in  cattle.  In  bovine  animals  the  early  diagnosis 
of  tuberculosis  is  clinically  impossible  in  the  majority  of  cases,  but  it  is  very 
important  from  the  point  of  view  of  prophylaxis  that  the  disease  should  be 
recognized  in  these  animals  in  its  very  earliest  stages. 

Tuberculous  cattle,  however  small  the  lesions  may  be,  react  to  the  inocula- 
tion of  0-30-0-40  c.c.  of  crude  tuberculin.  The  temperature  rises  l'5°-3°  C. 
Animals  free  from  tuberculosis  do  not  react  under  similar  conditions. 

The  method  of  diagnosis  is  as  follows. 


326  THE   TUBERCLE   BACILLUS 

1.  The  animal  to  be  tested  is  kept  quiet  and  its  temperature  taken  in  the 
rectum  the  day  before  as  well  as  on  the  day  on  which  it  is  to  be  tested. 

2.  Dilute  the  tuberculin. 

Crude  tuberculin,      -          -  1  c.c. 

Boiled  water  containing  0*5  per  cent,  carbolic  acid,        -  9     „ 

This  solution  will  not  keep  and  should  be  newly  prepared  for  each 
experiment. 

3.  With  the  usual  aseptic  precautions  inoculate  the  animal  sub-cutane- 
ously  in  the  neck  with  3-4  c.c.,  according  to  its  size,  of  the  diluted  tuberculin. 

4.  Take  the  temperature  12  Hours  after  inoculation  and  again  16  hours, 
20  hours  and  24  hours  after.     [Twelve  hours  is  too  late.     The  temperature 
should  be  taken  6,  9,  12,  15,  18;  24  and  finally  36  hours  after  inoculation.] 

Any  animal  which  during  that  period  shows  a  rise  of  temperature  of  1*4°  C. 
ought  to  be  regarded  as  tuberculous.  Animals  which  suffer  a  minimal  rise 
of  temperature  (0'5°-0'8°  C.)  are  healthy.  When  the  rise  of  temperature 
is  between  O8°  and  1'4°  C.  there  is  a  suspicion  of  tuberculosis  and  the  animal 
should  be  tested  again  a  month  later. 

Note. — The  tuberculin  reaction  though  of  great  diagnostic  value,  is  not  absolutely 
reliable.  In  severely  infected  animals  there  may  be  no  reaction.  On  the  other 
hand,  a  rise  of  temperature  of  1°  C.  is  not  sufficient  upon  which  to  base  a  diagnosis. 
A  few  cattle  which  showed  a  rise  of  2°  C.  were  subsequently  found  to  be  free  from 
tuberculosis  (Arloing,  Rodet  and  Courmont).  Infection  of  the  lung  with  echinococcus 
is  particularly  likely  to  give  the  temperature  reaction  in  cattle. 

B.  In  man. — Tuberculin  has  been  used  in  the  diagnosis  of  doubtful  cases 
of  the  disease  in  man. 

The  inoculation  of  tuberculin  is  always  attended  with  a  certain  amount  of 
danger  and  very  great  care  must  be  exercised  in  its  use.  In  any  case  the 
dose  injected  must  never  exceed  0*002  gram,  and  the  following  solution 
should  be  used. 

Crude  tuberculin,      -  4  c.c. 

Boiled  water  containing  0'5  per  cent,  carbolic  acid,       -         -         996     ,, 

One  c.c.  of  this  solution  contains  0'004  gram  of  crude  tuberculin  and  not 
more  than  a  fraction  of  1  c.c.  should  be  inoculated.  The  temperature  is 
taken  for  2  or  3  days  before  the  inoculation  and  every  8  hours  for  36  hours 
afterwards.  The  part  suspected  to  be  infected  must  be  carefully  watched, 
the  local  reaction  being  of  the  greatest  importance  from  the  point  of  view  of 
diagnosis.  In  patients  who  have  suffered  from  tuberculosis  for  a  very  long 
time,  small  doses  of  tuberculin  often  produce  no  temperature  reaction 
(Freymuth). 

As  to  the  amount  of  tuberculin  to  be  inoculated  to  obtain  a  reaction  observers 
differ.  There  are  three  methods  of  using  tuberculin  for  purposes  of  diagnosis. 

1.  Single  inoculation.— A  single  dose  of  0'5  c.c.   of  the  above  solution 
(0*002  gram  of  crude  tuberculin)  is  inoculated.     This  method  is  not  free 
from  danger. 

2.  Inoculation    of   increasing    doses. — German    observers,    among    whom 
tuberculin  is  largely  used  for  the  diagnosis  of  tuberculosis  in  man,  do  not 
hesitate  gradually  to  increase  the  amount  of  tuberculin  until  the  dose  inocu- 
lated is  very  considerable.     Generally  a  dose  of  0'5  mg.  of  crude  tuberculin 
is  inoculated  deeply  into  a  muscle  in  the  first  instance  and  then  gradually 
increasing  doses  of  1,  3,  6  and  even  10  or  20  mg.  every  3  or  4  days  until  the 
specific  reaction  is  obtained. 

In  soldiers  in  apparently  good  health,  Franz  found  that  tuberculin  in  doses  of 
1-3  mg.  set  up  a  reaction  in  about  64  per  cent,  of  the  men  and  when  the  dose  was 
increased  to  10  mg.  96  per  cent,  reacted. 


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TUBERCULIN  IN  DIAGNOSIS  327 


By  gradually  increasing  the  dose  of  tuberculin  a  non-specific  reaction  can 
[it  is  said]  be  produced  in  healthy  persons.  [But]  in  any  case  it  would  be 
most  unwise  to  inoculate  such  dangerous  doses. 

3.  Repeated  inoculation  of  small  doses.— Many  observers  advise  that  four 
five  small  doses,  O1-O2  mg.,  should  be  given  at  intervals  of  3  or  4  days. 

is  the  least  dangerous  method  and  the  one  to  be  adopted  to  avoid  the 
toward  results  which  so  frequently  follow  the  inoculation  of  tuberculin. 
Repeated  small  doses  lead  to  a  state  of  hypersensibility l  and  the  diagnosis 
can  be  made  without  running  the  risks  attendant  upon  the  use  of  increasing 
doses.  About  95  out  of  every  100  tuberculous  subjects  react  to  the  third 
or  fourth  inoculation. 

When  several  successive  inoculations  of  tuberculin  are  made  signs  of  inflammation 
appear  after  each  fresh  injection  around  the  sites  of  the  former  inoculations.  The 
lesions  produced  are  similar  to  those  set  up  [in  the  early  stages  of  an  infection]  by 
the  tubercle  bacillus  (Klingmueller). 

4.  Cuti-reaction. — Von  Pirquet  has  shown  that  when  tuberculin  is  inocu- 
lated through  scratches  on  the  skin  of  a  tuberculous  child,  in  nearly  every 
case  (save  in  acute  miliary  tuberculosis  and  tuberculous  meningitis)  a  small 
papule — occasionally  a  vesicle — appears  lasting  about  8  days  ;    at  first  it  is 
bright  red  but  subsequently  becomes  darker  in  colour.     This  reaction  is  of 
great  diagnostic  value  in  the  early  years  of  life.     Older  children  -often,  and 
adults  nearly  always,  react  even  when  tuberculosis  cannot  be  demonstrated 
clinically  (which  calls  to  mind  the  fact  that  post  mortem  nearly  all  adults  show 
lesions  of  tuberculosis).     In  cachectic  individuals  the  reaction  often  fails. 

To  effect  the  reaction  make  four  small  scarifications,  not  deep  enough  to  draw 
blood,  on  the  skin  of  the  outer  and  upper  part  of  the  arm  and  cover  the  lower  three 
with  a  small  drop  of  diluted  tuberculin  (p.  326).  (Tubes  containing  tuberculin  for 
the  cuti-reaction  can  be  purchased  at  the  shops.)  The  vaccination  marks  should 
be  about  2  cm.  apart.  The  upper  mark  which  has  not  been  treated  with  tuberculin 
serves  as  a  control.  When  the  reaction  is  positive,  a  swelling  begins  to  appear 
about  48  hours  after  inoculation. 

H.  Vallee  has  shown  that  von  Pirquet's  cuti-reaction  is  of  value  as  a 
diagnostic  test  for  tuberculosis  in  the  lower  animals  (cattle,  horses  and 
guinea-pigs).  While  healthy  animals  do  not  react,  tuberculous  animals 
show,  about  36-48  hours  after  the  inoculation,  an  oedematous  infiltration 
of  the  vaccination  mark  with  a  painful  grey-red  swelling.  The  reaction 
occurs  in  healthy  animals  which  have  been  previously  treated  with  a  sub- 
cutaneous inoculation  of  tuberculin  (this  confirms  Klingmueller's  phenomenon 
described  above). 

5.  Intra-dermo  reaction.— This  method  of  diagnosis  is  recommended  by 
Martoux  but  does  not  seem  to  offer  any  advantages  over  the  cuti-reaction. 
The  reaction  consists  in  inoculating  a  drop  of  tuberculin  into  the  skin  with  a 
fine  needle.     Calmette  advises  a  1  in  5000  solution  of  dry  tuberculin  preci- 
pitated with  alcohol.     In  persons  affected  with  tuberculosis  a  red,  or  bright 
pink,  cedematous  infiltration  surrounded  by  a  more  or  less  extensive  area  of 
erythema  is  seen  about  24  hours  after  the  inoculation. 

6.  Ophthalmo-reaction. — Calmette  and  Wolff-Eisner  have  shown  that  the 
instillation  of  a  small  amount  of  tuberculin  into  the  eye  produces  in  persons 
affected  with  tuberculosis  a  very  marked  congestion  of  the  palpebral  conjunc- 
tiva.    The  test  is  easily  performed  and  is  very  delicate  and,  provided  it  is 
not  used  when  any  lesion  of  the  eye  is  present  nor  in  old  people,  is  quite  free 
from  danger. 

[x  Repeated  injections  of  large  doses  sometimes  diminish  the  reacting  power  of  the 
tissues  in  animals.] 


328  THE   TUBERCLE   BACILLUS 

Calmette  says  that  crude  tuberculin  should  never  be  used  for  the  ophthalmo- 
reaction  because  the  glycerin  present  in  solution  acts  as  an  irritant.  He 
uses  a  recently  made  1  per  cent,  solution  of  dried  tuberculin  (twice  preci- 
pitated with  95  per  cent,  alcohol)  in  distilled  water.  One  drop  of  the  tuber- 
culin is  instilled  into  one  eye.  Three  to  five  hours  later,  in  all  tuberculous 
persons,  there  is  congestion  of  the  inner  end  of  the  palpebral  conjunctiva, 
and  more  or  less  oedema  and  swelling  of  the  caruncle  which  is  covered 
with  a  slight  fibrinous  exudate.  Lachrymation  occurs  and  there  is  a  little 
discomfort  but  no  pain.  The  reaction  reaches  its  maximum  in  6-10  hours, 
and  after  18  hours  in  children  and  24  hours  in  the  adult  the  signs  subside 
and  disappear. 

In  persons  not  infected  with  tuberculosis  instillation  occasionally  produces 
a  little  redness  but  there  is  never  lachrymation  nor  any  fibrinous  exudate. 

The  advantage  of  the  ophthalmo-reaction  is,  according  to  Calmette,  that 
it  indicates  the  presence  of  active  lesions  only  and  as  a  means  of  diagnosis 
is  of  more  value  than  the  cuti-reaction.  A  negative  reaction  is  sufficient  to 
exclude  a  tuberculous  infection  except  in  tuberculous  persons  suffering  from 
cachexia  who  have  lost  the  power  of  reacting.  Persons  who  have  recovered 
from  a  tuberculous  infection  give  a  negative  reaction.  [Other  observers 
confirm  Calmette,  finding  that]  with  rare  exceptions  (some  cases  of  enteric 
fever,  for  instance,)  a  positive  reaction  indicates  an  active  tuberculous  infection. 

[7.  Percutaneous  reaction. — Moro  rubs  a  lanolin  ointment  containing  50  per 
cent,  of  Koch's  old  tuberculin  into  the  skin  of  the  chest  or  abdomen  over  an 
area  of  about  three  square  inches.  From  1-2  days  after  the  application 
numerous  small  red  papules  appear  on  the  anointed  surface  in  tuberculous 
individuals.  The  eruption  is  transitory  and  the  reaction  quite  painless. 
The  results  are  very  comparable  with  those  given  by  von  Pirquet's  method.] 

C.  Tuberculins  TA,  TO,  and  TR. 

Koch  investigated  a  number  of  complex  products  derived  in  various  ways 
from  cultures  of  the  tubercle  bacillus.  These  are  known  as  tuberculin  TA, 
tuberculin  TO,  and  tuberculin  TR. 

1.  Tuberculin   TA    (alkaline   tuberculin). — Tubercle   bacilli    separated    by 
filtration  from  a  virulent  culture  are  treated  with  a  10  per  cent,  solution  of 
soda  and  after  3  days'  exposure  at  room  temperature  the  bacilli  are  killed 
and  the  liquid  can  be  filtered  through  paper.     After  neutralization  the  filtrate 
is  clear,  slightly  yellowish  and  contains  numerous  dead  bacilli.     Inoculation 
of  the  filtrate  produces  results  similar  to,  but  rather  more  persistent  than, 
ordinary  tuberculin,  and  often  leads  to  the  formation  of  an  abscess  contain- 
ing sterile  pus.     After  filtering  through  a  bougie  TA  gives  results  identical 
with  ordinary  tuberculin. 

2.  Tuberculin  TO  and  tuberculin  TR. — Bacilli  from  a  young,  virulent  culture 
are  dried  in  vacuo  in  the  dark  and  then  rubbed  up  in  an  agate  mortar  for 
a  long  time.     This  is  a  dangerous  proceeding  and  should  be  carried  out  with 
the  utmost  care.     The  powder  so  obtained  is  mixed  with  distilled  water  and 
the  emulsion  centrifuged  for  40  or  45  minutes  (4000  revolutions  a  minute). 
Two  layers  are  formed  ;  the  upper  (obere),  fluid  and  opalescent  and  containing 
no  bacilli,  is  decanted  off  and  forms  tuberculin  TO. 

The  muddy  lower  layer  is  dried,  rubbed  up  again,  mixed  with  disti  led 
water  and  centrifuged.  The  residue  from  the  centrifuging  is  treated  in  the 
same  way  and  the  operation  is  repeated  several  times.  Finally  the  liquids 
poured  off  after  each  centrifuging  are  mixed  together  and  form  tuberculin  TR 
[Riickstand]. 

Tuberculin  TO  is  very  different  from  tuberculin  TR. 


MARAGLIANO'S   TUBERCULIN  329 

Tuberculin  TO  is  not  altered  by  the  addition  of  50  per  cent,  of  glycerin  : 
its  properties  are  almost  identical  with  those  of  ordinary  tuberculin  and  its 
immunizing  properties  are  nil  or  very  little  marked. 

Tuberculin  TR  gives  a  flocculent,  white  precipitate  on  the  addition  of  50 
per  cent,  of  glycerin.  Its  characters  are  unaltered  by  the  addition  of  20  per 
cent,  of  glycerin  which,  on  the  other  hand,  preserves  it. 

According  to  Koch  tuberculin  TR  has  distinct  immunizing  properties. 
Repeated  inoculation  of  small  doses  into  the  human  subject  confers  an  im- 
munity against  ordinary  tuberculin  and  TO,  as  well  as  against  itself.  These 
statements  have  not  been  confirmed  (Bounhiol)  ;  tuberculin  TR,  whatever 
Koch  may  have  said,  appears  to  have  no  power  of  arresting  tuberculosis. 
In  tuberculous  persons,  a  reaction  similar  to  that  produced  by  ordinary 
tuberculin  occurs  but  very  inconstantly.  It  appears  to  be  less  dangerous 
than  ordinary  tuberculin,  but,  on  account  of  the  irregularity  of  its  effects, 
it  cannot  be  used  for  purposes  of  diagnosis. 

D.  Maragliano's  tuberculin. 

This  is  a  watery  tuberculin  obtained  by  maceration  of  tubercle  bacilli. 
Recover  the  bacilli  from  a  glycerin-broth  culture,  add  to  them  a  volume 
of  distilled  water  equal  to  the  volume  of  the  culture'  fluid  and  heat  to  a 
temperature  of  95°-100°  C.  for  50  hours  or  so.  Then  evaporate  on  a  water 
bath  to  one-tenth  its  original  volume  and  filter  through  filter  paper. 

The  filtrate  has  the  same  properties  as  Koch's  old  tuberculin  and  is  said 
to  possess  vaccinating  properties.  Doses  of  5  c.c.  are  fatal  to  healthy  guinea- 
pigs  weighing  500  grams.  Tuberculous  guinea-pigs  succumb  to  the  inocula- 
tion of  0'10-0'20  c.c.  of  this  tuberculin.  • 

When  precipitated  with  alcohol,  Maragliano's  tuberculin  yields  a  powder 
which  kills  guinea-pigs  in  doses  equivalent  to  25,o00th  of  their  weight  and 
rabbits  in  amounts  corresponding  to  3a,o00th  of  their  weight. 

E.  Toxalbumin. 

The  different  toxic  products  which  have  just  been  studied  are  contained 
within  the  bacilli — endotoxins — and  to  extract  them  the  bacilli  have  to  be 
killed  by  heat  or  destroyed  by  trituration. 

Maragliano,  Bezancon  and  Gouger  have  shown  that  the  tubercle  bacillus 
produces  a  diffusible  toxin  which  passes  into  the  medium  in  which  the  bacilli 
are  growing.  This  toxin  is  of  the  nature  of  a  toxalbumin  :  it  is  destroyed 
by  heating  it  to  100°  C.  and  by  prolonged  exposure  to  light.  It  is  prepared 
by  filtering  a  glycerin-broth  culture  through  porcelain  and  concentrating  the 
filtrate  to  one-tenth  its  original  volume  in  vacuo  at  55°  C. 

The  product  differs  absolutely  from  the  tuberculins  :  it  is  more  toxic  than 
the  latter  and  inoculated  into  animals  it  never  produces  a  rise  of  temperature 
even  in  non-fatal  doses  :  in  fatal  doses  the  animals  die  with  a  sub-normal 
temperature. 

Denys  uses  a  porcelain-filtered  glycerin-broth  culture  of  the  tubercle  bacillus 
in  the  treatment  of  tuberculosis. 

Beraneck  prepares  a  toxin-broth  by  growing  the  tubercle  bacillus  in  a 
maceration  of  veal  made  in  the  cold  to  which  0*5  per  cent,  salt  and  5  per 
cent,  glycerin  are  added  before  sterilization.  .By  the  time  that  the  surface 
is  covered  with  a  pellicle,  the  medium,  originally  alkaline,  has  become  acid. 
It  is  then  made  alkaline  with  lime  water,  filtered  through  a  Chamberland 
bougie,  and  the  filtrate  evaporated  in  vacuo  to  one-tenth  its  original  volume. 

Beraneck  recommends  for  the  treatment  of  tuberculosis  the  use  of  this  toxin  broth 
diluted  with  an  equal  volume  of  an  acid  extract  of  the  bodies  of  bacilli  containing 


330  THE  TUBERCLE   BACILLUS 

endotoxin.  This  extract,  or  acido-toxin,  is  made  by  macerating  tubercle  bacilli  in  a 
10  per  cent,  aqueous  solution  of  ortho-phosphoric  acid  for  2  hours  at  60°  C.,  neutraliz- 
ing the  product,  filtering  and  diluting  nineteen  times  with  water  (1  in  20  solution  of 
endotoxin). 

3.  Vaccination. 
A.  Laboratory  animals. 

(i)  Grancher  and  Martin. — Rabbits  are  inoculated  with  avian  tubercle 
bacilli  which  have  been  grown  on  artificial  media  for  prolonged  periods.  By 
using  successively  less  and  less  attenuated  cultures  they  have  succeeded  in  a 
few  cases  in  producing  a  certain  degree  of  immunity  in  the  animals. 

(ii)  Hericourt  and  Richet  sterilize  cultures  of  avian  bacilli  by  heating 
them  several  times  to  80°  C.  and  then  inoculate  them  into  rabbits  in  doses  of 
10-20  c.c.  This  method  has  enabled  them  to  confer  immunity  on  a  few 
animals. 

(iii)  Courmont  and  Dor  filter  glycerin-broth  cultures  and  inoculate  the 
filtrate  into  rabbits  at  the  same  time  that  or  before  they  inoculate  a  tuber- 
culous virus.  With  bacilli  of  avian  origin  they  succeeded  in  producing 
immunity  twice  in  four  experiments  but  they  failed  with  the  bacillus  of 
human  origin. 

(iv)  In  the  foregoing  experiments,  attempts  to  vaccinate  guinea-pigs  had 
failed.  E.  Levy  immunizes  guinea-pigs  by  inoculating  them  with  a  tubercle 
bacillus  [which  he  states  to  have  been]  attenuated  by  being  kept  in  glycerin 
(p.  322). 

Two  guinea-pigs  are  inoculated,  one  into  the  peritoneal  cavity,  the  other  sub- 
cutarftously,  with  a  slightly  opalescent  emulsion  of  tubercle  bacilli  which  has  been 
kept  in  80  per  cent,  glycerin  for  6  days  in  the  incubator  at  37°  C.  When  they  have 
recovered  from  the  first  inoculation  they  are  again  inoculated  on  successive  occasions 
with  bacilli  which  have  been  kept  in  glycerin  for  4,  3  and  2  days.  When  they  have 
completely  recovered  they,  as  well  as  two  control  animals,  are  inoculated  with  a 
tubercle  bacillus  of  standard  virulence.  All  the  animals  develop  an  abscess  at  the 
site  of  inoculation  but  at  the  end  of  about  4  weeks  the  lesion  in  the  case  of  the 
vaccinated  guinea-pigs  has  healed  while  in  the  controls  the  disease  has  spread  to 
the  glands.  Towards  the  end  of  the  third  month  the  controls  are  suffering  from  a 
generalized  tuberculosis  (liver,  spleen  and  lungs)  while  the  most  minute  search 
fails  to  reveal  any  trace  of  tuberculosis  in  the  vaccinated  animals  killed  at  the  same 
time. 

B.  Cattle. 

(i)  Von  Behring  succeeded  in  vaccinating  young  bovine  animals  (healthy 
calves  under  one  year  old)  by  inoculating  them  intra-venously  with  living 
attenuated  bacilli  of  human  origin,  non-virulent  for  cattle  (bovo-vaccin). 

The  bacillus  used  by  von  Behring  in  his  experiments  was  a  human  tubercle  bacillus 
which  had  been  in  artificial  culture  for  8  years  and  had  lost  much  of  its  original 
virulence  (vide  p.  322,  attenuation  of  tubercle  bacilli).  A  five- week-old  culture  of 
this  bacillus  oh  glycerin -serum  could  be  inoculated  with  impunity  into  the  veins  of 
a  calf  in  doses  of  0'005  gram.  A  first  injection  of  0*001  gram  provoked  no  reaction 
and  was  followed  by  several  other  inoculations  with  increasing  doses  at  intervals  of 
several  weeks.  Animals  treated  in  this  way  were  finally  able  to  resist  doses  of 
bacilli  of  bovine  type  which  were  fatal  to  the  control  animals. 

Behring  has  now  modified  his  original  procedure.  He  first  inoculates,  intra- 
venously, 0'004  gram  of  a  glycerin-agar  culture  of  a  bacillus  of  human  origin  com- 
pletely dried  in  vacua  at  the  ordinary  temperature  (the  bacilli  are  ground  up  in  a 
mortar  and  emulsified  in  4  c.c.  of  a  1  per  cent,  saline  solution).  A  month  later 
the  animal  is  similarly  inoculated  with  0'01-0'02  gram  of  the  same  culture. 

Animals  treated  in  this  way  and  exposed  to  contagion  or  inoculated  with  a  virulent 
bacillus  of  bovine  origin  never  develop  any  tuberculous  lesion.  And,  further, 
when  tested  with  tuberculin  a  year  after  immunization  they  always  fail  to  react. 


VACCINATION   OF   CATTLE  331 

With  other  observers  this  method  of  vaccination  has  always  given  favour- 
able results,  but  complete  immunity  has  not  been  attained. 

Vallee  and  Rossignol  undertook  a  series  of  control  experiments  on  twenty  calves 
each  about  5  months  old  which  had  not  reacted  to  tuberculin.  These  animals  were 
first  inoculated  in  the  jugular  vein  with  4  mg.  of  dried  tubercle  bacilli  (bovo-vaccin 
of  von  Behring)  and  3  months  later  with  20  mg.  of  similar  bacilli.  They  were  tested 
by  ultra- venous  and  sub-cutaneous  inoculation  and  by  being  kept  in  contact  with 
animals  suffering  from  open  tuberculous  lesions.  From  these  experiments,  Vallee 
and  Rossignol  conclude  that  von  Behring' s  method  of  vaccination  is  harmless  to 
animals  protected  from  sources  of  accidental  infection  during  the  period  of  immuniza- 
tion and  for  6  weeks  afterwards :  that  the  vaccination  confers  considerable  but  not 
absolute  powers  of  resistance  to  the  most  severe  methods  of  infection  and  that  the 
vaccinated  animals  are  able  to  resist  for  some  months  spontaneous  infection  which 
might  have  been  expected  to  arise  from  prolonged  contact  with  infected  animals. 

Von  Behring's  method  of  vaccination  should  be  performed  on  animals 
less  than  3  months  old.  In  calves  more  than  a  year  old  the  vaccinating  process 
occasionally  sets  up  a  violent  reaction  and  is  dangerous  to  life.  The  severity 
of  this  reaction  seems  to  depend  upon  an  anterior  infection  with  tuberculosis 
and  it  is  well  known  that  in  cattle  the  younger  the  animal  the  more  rare  the 
disease.  The  resulting  immunity  is  especially  noticeable  when  the  animals 
are  tested  by  intra-venous  inoculation  but  is  only  effective  for  a  few  months 
when  tested  by  feeding  the  animals  with  infected  food  stuffs  or  keeping  them 
in  contact  with  cattle  suffering  from  open  tuberculous  lesions. 

(ii)  With  a  view  to  getting  more  constant  results  and  a  more  efficient 
method  of  vaccination  some  observers  have  experimented  with  living  and 
virulent  bacilli  of  the  human  type.  Experience  has  shown  that  the  immunity 
produced  in  cattle  by  the  inoculation  of  living  cultures  runs  parallel  with  the 
virulence  of  the  tubercle  bacillus  used  in  the  experiments,  the  higher  the 
virulence  of  the  bacillus  the  greater  the  degree  of  immunity  produced. 

[A.  S.  Griffith  and  F.  Griffith  (English  Commission)  investigated  the  produc- 
tion of  immunity  in  calves  by  the  inoculation  of  living  tubercle  bacilli.  Twelve 
calves  were  inoculated  sub-cutaneously  or  intra-venously,  ten  with  human 
and  two  with  bovine  tubercle  bacilli  and  were  subsequently  tested  as  to  their 
resistance  by  the  sub -cutaneous  inoculation  of  a  large  dose  (50  mg.)  of  bovine 
tubercle  bacilli.  Nine  of  the  calves  "  had  their  resistance  so  far  increased 
that  50  mg.  of  bovine  tubercle  bacilli  were  unable  to  set  up  in  them  pro- 
gressive tuberculosis."  Of  the  remaining  three  two  died  of  acute  tuberculosis 
and  the  third  when  killed  showed  general  tuberculosis  but  in  a  less  severe 
degree  than  in  any  of  the  four  control  animals.  Thus  "  by  the  inoculation 
of  large  doses  of  human  or  small  doses  of  bovine  tubercle  bacilli,  the  resistance 
of  a  calf  can  be  raised  sufficiently  to  protect  it  against  the  inoculation  of  a 
dose  of  bovine  tubercle  bacilli  capable  of  setting  up  a  severe  and  fatal  tuber- 
culosis in  a  calf  not  so  protected  "  ;  but  "  this  degree  of  resistance  is  not 
always  produced."  There  was  no  evidence  to  show  that  vaccination  with 
bovine  tubercle  bacilli  produced  a  higher  degree  of  immunity  than  vaccination 
with  human  tubercle  bacilli.  ] 

Bacilli  killed  by  heat  are  devoid  of  vaccinating  properties. 

But  vaccination  with  an  active  virus  is  not  free  from  danger ;  inoculation 
of  living  bacilli  of  the  human  type  may  [it  is  said]  set  up  latent  lesions 
which  can  be  re-awakened  and  constitute  a  permanent  menace  of  re-infection ; 
and  finally,  it  is  a  danger  to  the  consumer  of  the  meat  [and  milk]. 

[A.  S.  Griffith  (English  Commission)  found  that  if  milking  cows  are  "  inocu- 
lated sub-cutaneously  or  intra-venously  with  tubercle  bacilli  of  relatively 
slight  virulence  such  bacilli  quickly  appear  in  their  milk  and  may  continue 


332  THE   TUBERCLE   BACILLUS 

to  be  eliminated  therein  for  long  periods."  It  is  obvious  from  these  experi- 
ments that  the  vaccination  of  milch  cows  with  living  bacilli  is  undesirable. 
Other  experiments  by  this  observer  also  seem  to  indicate  that  this  method 
of  vaccination  is  not  free  from  danger.  In  "  seven  out  of  eleven  heifers 
(ages  6-10  months)  tubercle  bacilli  of  various  types  which  had  been  inocu- 
lated in  large  doses  into  the  sub-cutaneous  tissues  had  found  their  way  into 
the  milk  sinuses  of  the  undeveloped  mamma  "  and  in  four  cases  at  least 
appeared  to  have  undergone  multiplication  since  their  arrival  there.  There 
is  therefore  reasonable  probability  that  the  first  milk  of  some  of  these  animals 
would  have  contained  living  tubercle  bacilli. 

[Immunization  with  living  tubercle  bacilli,  whether  human  or  bovine,  is 
therefore  not  free  from  risk.] 

Thomassen  injected  2-3  eg.  of  fresh  tubercle  bacilli  from  an  human  source  grown 
on  glycerin-potato  into  the  jugular  vein  of  calves  ;  as  a  rule,  the  animals  were 
resistent  to  a  subsequent  inoculation  of  bacilli  of  bovine  origin  but  the  animals 
might  succumb  to  the  vaccinating  inoculation  and  in  those  that  recovered  it  was 
possible  that,  the  disease  might  subsequently  recur.  Thomassen  preferred  to  give 
increasing  doses  (1  mg.  at  the  first  inoculation,  10  mg.  a  month  later  and  finally 
20  mg.).  The  results  were  good  but  not  constant.  It  was  not  uncommon  to  find 
that  in  vaccinated  animals  which  had  failed  to  react  to  the  test  inoculation,  the 
bronchial  glands,  though  normal  in  appearance  when  the  animal  was  killed  a  few 
weeks  later,  produced  'tuberculosis  on  inoculation  into  guinea-pigs.  [Cobbett  (for 
the  English  Commission)  found  that  human  tubercle  bacilli  inoculated  into  calves 
remained  alive  for  prolonged  periods,  in  lesions  so  minute  as  to  be  hardly  visible.] 

Hutyra  had  good  results  with  a  similar  method  (inoculation  of  a  recent 
potato  culture  of  tubercle  bacilli  of  human  origin). 

Baumgarten  obtained  very  favourable  results  by  simple  sub-cutaneous 
inoculation  of  human  tubercle  bacilli  of  standard  virulence.  According  to 
the  author  there  was  a  non-specific,  local,  inflammatory  lesion. 

(iii)  Koch  and  others  immunized  calves  by  inoculating  them  with  an 
attenuated  bacillus  of  bovine  origin.  The  inoculations  were  made  into  the 
jugular  vein,  10  mg.  on  the  first  occasion  and  25  mg.  3  weeks  later. 

It  did  not  appear  to  be  a  true  vaccination  but  a  more  or  less  brief  increase  of 
resistance  to  the  action  of  the  bovine  type  of  bacillus.  An  heifer  which  had  been 
vaccinated  did  not  react  to  the  test  inoculation  and  remained  in  apparent  good 
health  but  died  14  months  later  while  suckling  a  calf.  Lactation  would  appear  to 
re-awaken  a  latent  infection  (Pepere). 

[Pregnancy  appears  to  render  the  sex-organs  peculiarly  susceptible  to 
tuberculosis.  Cobbett  (for  the  English  Commission)  records  that  three  out  of 
six  heifers  inoculated  when  pregnant  with  bovine  tubercle  bacilli  developed 
tuberculosis  of  the  uterus.  In  none  of  the  three  was  the  generalized  disease 
very  acute,  and  in  one  (in  which  the  mammary  gland  also  was  affected)  there 
was  very  little  tuberculosis  elsewhere.  The  non-pregnant  uterus,  he  con- 
tinues, has  never  been  found  affected  in  calves  suffering  from  general  tubercu- 
losis however  severe.] 

Klemperer  endeavoured  to  treat  persons  suffering  from  tuberculosis  by 
inoculating  them  with  tubercle  bacilli  of  bovine  type.  He  is  said  to  have 
noticed  improvement  in  the  condition  of  the  patients  but  his  experiments 
are  not  conclusive.  Similarly  the  inoculation  of  bacilli  of  the  human  type 
into  tuberculous  cows  appeared  to  have  no  curative  action. 

(iv)  Vallee  succeeded  in  vaccinating  cattle  against  the  effects  of  the  injec- 
tion of  tuberculin  and  in  conferring  some  degree  of  immunity  against  living 
cultures  by  inoculating  them  intra-venously  with  dead  tubercle  bacilli  from 
which  the  fat  had  been  extracted. 


IMMUNIZATION  BY  FEEDING  333 

The  bacilli  were  rapidly  washed  in  distilled  water,  drained  and  dried  for  several 
days  in  vacuo  over  sulphuric  acid,  then  placed  in  a  flat-bottomed  flask  with  pure 
petroleum  ether  and  glass  balls.  The  flask  was  placed  in  a  shaking  apparatus  and 
the  contents  shaken  for  60  hours. 

The  bacilli  had  then  lost  their  acid-fast  properties.  The  emulsion  was  dried  in  a 
desiccator  to  remove  all  traces  of  the  ether  from  the  bodies  of  the  bacilli. 

Tubercle  bacilli  treated  in  this  manner  behave  like  a  very  powerful  tuber- 
culin. They  kill  guinea-pigs  in  doses  of  70  mg.  When  inoculated  several 
times  in  doses  of  from  25-100  mg.  into  the  jugular  vein  they  [are  said  to] 
render  young  calves  to  a  certain  degree  immune  to  the  bacillus  of  bovine 
type.  Horses  and  cattle  rendered  immune  to  the  inoculation  of  these  bacilli 
cease  to  react  to  the  intra-venous  inoculation  of  the  various  tuberculins. 

(v)  Animals  vaccinated  by  inoculating  them  intra-venously  show  them- 
selves very  slightly  immune  to  intestinal  infection — a  mode  of  infection 
particularly  common  in  nature.  Behring,  Calmette  and  Guerin,  Roux  and 
Vallee  have  tried  to  immunize  animals  by  feeding  them  with  tubercle  bacilli 
and  have  met  with  some  success.  The  method  is  only  practicable  in  the 
case  of  young  cattle  and  the  resulting  immunity  is  particularly  efficient 
against  intestinal  infection.  Although  the  immunity  is  only  relative  the 
method  gives  better  results  than  intra-venous  inoculation  :  the  animals 
remain  uninfected  for  a  year  when  kept  with  cattle  suffering  from  open 
lesions. 

Two  young  calves  were  fed  at  intervals  of  45  days  with  5  and  25  grams  of  tubercle 
bacilli  of  the  human  type.  Four  months  later  failing  to  react  to  tuberculin  they 
were  fed  with  0'05  gram  of  freshly  isolated  bacilli  of  the  bovine  type  :  32  days  later 
they  failed  to  react  to  tuberculin  while  two  controls  reacted  in  the  ordinary  way. 

Vallee  also  immunized  a  young  calf  by  feeding  it,  through  an  cesophageal  sound, 
on  two  occasions,  when  it  was  2  days  and  90  days  old  respectively,  with  20  eg.  of  a 
well-made  emulsion  of  a  tubercle  bacillus  of  equine  origin  which  was  only  slightly 
virulent  for  guinea-pigs. 

Roux  and  Vallee,  Calmette  and  Guerin  have  shown  that  small  doses  of  virulent 
bacilli  of  the  bovine  type  when  introduced  into  the  alimentary  canal  of  the  calf 
are  absorbed  into  the  mesenteric  glands  and  give  rise  to  a  substantial  immunity  in 
the  animal.  A  certain  amount  of  risk  attaches  to  this  method  of  immunization 
(Vallee). 

(vi)  For  the  purpose  of  producing  immunity  Arloing  uses  homogeneous 
cultures  of  bacilli  of  the  human  type  (vide  infra)  which  have  lost  much  of 
their  capacity  of  producing  tubercles  ;  by  submitting  them  to  gradually 
increasing  temperatures  Arloing  was  able  to  grow  them  at  43°-44°  C.  These 
cultures  vaccinate  calves  and  appear  to  act  as  a  true  pasteurian  vaccine. 

C.  Vaccination  with  a  virus  of  chelonian  origin. 

Friedmann  showed  that  a  bacillus  which  he  had  recovered  from  a  tortoise 
(p.  297)  produced  when  inoculated  beneath  the  skin  of  a  guinea-pig  a  typical 
localized  tuberculous  focus  which  soon  completely  healed  while  the  animal 
never  showed  any  sign  of  generalized  tuberculosis.  Further,  guinea-pigs 
treated  in  this  way  resisted  the  inoculation  of  a  dose  of  bacilli  of  the  human 
type  which  killed  control  animals  in  4-6  weeks. 

In  vaccinated  guinea-pigs  the  inoculation  of  a  virus  of  human  origin  gave  rise 
to  a  transitory  swelling  of  the  glands  and  to  a  caseo- purulent  tuberculous  focus 
which  healed  and  left  no  trace  of  the  injury :  when  the  animals  were  killed 
about  3  months  later  no  lesion  was  found :  it  is  true  that  small  whitish  points  were 
seen  in  the  internal  organs  but  these  were  in  no  way  suggestive  of  true  tubercles 
and  similar  lesions  are  found  in  animals  immune  to  tuberculosis  or  vaccinated  in 
various  ways  against  the  disease  (Koch,  von  Behring,  Neufeld,  Thomassen,  and 
others). 


334  THE   TUBERCLE   BACILLUS 

The  method  is  available  for  the  immunization  of  bovine  animals  against 
tubercle  bacilli  of  the  bovine  type.  Intra-venous  inoculation  of  calves  with 
a  bacillus  of  chelonian  origin  [is  said  to  ]  produce  a  lasting  immunity  against 
bacilli  of  the  bovine  type.  The  method  might  also  be  applicable  to  the  treat- 
ment of  animals  suffering  from  tuberculosis.  Libbertz  and  Ruppel  have 
not  been  able  to  confirm  these  results. 

Conversely,  Friedmann  succeeded  in  vaccinating  tortoises  against  bacilli 
of  chelonian  origin  by  inoculating  them  with  a  virus  of  the  human  type. 

Moeller,  carrying  out  similar  researches,  succeeded  in  infecting  blind-worms 
with  human  tubercle  bacilli  and  for  the  purpose  used  as  a  vaccine  a  bacillus 
of  the  human  type  which  had  been  passed  through  a  series  of  blind-wTorms. 
Moeller  did  not  hesitate  to  practise  his  method  of  vaccination  on  himself. 
On  three  separate  occasions  he  inoculated  himself  intra-venously  with  a 
culture  of  the  bacillus  from  the  lesions  in  the  blind-worm  and  a  month  after 
his  last  vaccination  he  was  inoculated  intra-venously  with  an  emulsion  of 
virulent  bacilli  of  the  human  type  which  was  rapidly  fatal  to  guinea-pigs. 
This  inoculation  produced  merely  a  transitory  loss  of  weight  without  any 
disturbance  of  health  a  year  afterwards. 

4.  Serum  therapy. 

Attempts  at  serum  therapy  in  tuberculosis  have  up  till  the  present  given 
no  conclusive  results. 

(i)  Bichet  and  Hericourt  by  inoculating  rabbits  with  dog-serum  before 
infecting  them  with  tubercle  bacilli  have  been  able  to  delay  the  course  of 
infection  in  some  of  the  inoculated  animals.  Unfortunately,  the  success  of 
the  experiments  was  very  relative  and  inconstant.  Bertin  and  Picq,  experi- 
menting with  inoculations  of  goat  serum,  obtained  similar  results. 

(ii)  Von  Behring  and  Niemann  also  failed  with  the  serum  of  animals  treated 
with  tuberculin. 

(iii)  Bernheim  tried  the  blood  of  animals  inoculated  with  filtered  but 
unheated  cultures  of  tubercle  bacilli.  His  experiments  were  unsuccessful. 
The  results  obtained  by  Babes  and  Broca  were  no  more  encouraging. 

(iv)  Maragliano  obtained  a  serum  of  obvious  antitoxic  properties.  He 
injected  animals  with  increasing  doses  of  a  mixture  of  three  parts  of  ordinary 
tuberculin  and  one  part  of  an  extract  of  porcelain-filtered  unheated  cultures 
(p.  329).  The  treatment  was  continued  for  6  months  and  when  3  weeks  had 
elapsed  since  the  last  inoculation  the  animals  were  bled.  In  vitro.,  the  serum 
destroyed  the  toxic  properties  of  tuberculin  and  protected  guinea-pigs  against 
this  poison  :  1  c.c.  of  the  serum  protected  a  healthy  guinea-pig  against  a 
fatal  dose  of  tuberculin  :  2-4  c.c.  rendered  a  tuberculous  guinea-pig  capable 
of  standing  without  harm  a  dose  of  tuberculin  which  in  the  ordinary  way 
would  have  killed  it  in  a  few  hours.  Experiments  to  determine  whether 
the  serum  protected  healthy  animals  against  infection  with  the  tubercle 
bacillus  were  not  conclusive. 

(v)  Marmorek  for  the  treatment  of  tuberculosis  suggests  the  use  of  a 
serum  obtained  by  inoculating  horses  with  a  special  toxin  (a  filtered  culture 
of  bacilli  grown  on  a  leucotoxic  calf  serum  containing  glycerin-liver-broth). 

(vi)  Lannelongue,  and  Achard  and  Gaillard  found  that  the  serum  of  asses 
possessed  therapeutic  properties  after  the  animals  had  been  treated  by 
inoculation  with  a  toxin  extracted  from  the  bacilli  by  heating  in  water  at 
120°  C.,  precipitating  with  acetic  acid  and  redissolving  the  precipitate  in 
sodium  carbonate. 

(vii)  Baumgarten  and  Hegler  after  vaccinating  a  bovine  animal  with 
bacilli  of  the  human  type  were  able  to  inoculate  bacilli  of  the  bovine  type 


SERUM  THERAPY— AGGLUTINATION  335 

on  five  successive  occasions  without  producing  any  appreciable  reaction. 
The  tuberculin  test  was  negative  and  the  serum  of  this  animal  inoculated 
prophylactically  into  a  calf  (82  c.c.  sub-cutaneously  in  a  fortnight)  protected 
the  latter  against  a  sub-cutaneous  inoculation  of  bacilli  of  the  bovine  type 
while  two  controls  similarly  inoculated  with  the  test  culture  became  infected. 
The  passively  immunized  calf  when  killed  6  months  afterwards  showed  no 
tuberculous  lesion.  As  a  curative  agent  this  serum  is  without  effect. 

(viii)  Vallee  treats  horses  by  inoculating  them  first  with  progressively 
increasing  doses  of  bacilli  of  equine  origin  then  with  bacilli  of  human  origin. 
The  serum  of  such  animals  exhibits  no  agglutinating  properties  but  contains 
immune  bodies  (sensibilisatrices)  and  has  distinct  therapeutic  properties  in 
the  treatment  of  bovine  tuberculosis. 

(ix)  The  serum  of  bovine  animals,  guinea-pigs  and  pigs  vaccinated  by 
Friedmann  with  the  chelonian  bacillus  has  proved  itself  in  the  case  of  the 
guinea-pig  to  possess  undoubted  prophylactic  properties.  While  control 
guinea-pigs  died  of  generalized  tuberculosis,  the  treated  guinea-pigs  killed 
at  the  same  time  only  showed  insignificant  lesions. 

5.  Agglutination. 

1.  The  serum  diagnosis  of  tuberculosis  with  ordinary  cultures,  consisting 
as  they  do  of  bacilli  massed  together  to  form  films  or  scales,  is  quite  impossible. 
Arloing  and  Courmont  have  described  a  method  of  obtaining  homogeneous 
cultures  which  are  quite  suitable  for  the  demonstration  of  the  phenomena 
of  agglutination. 

Arloing's  homogeneous  cultures  are  obtained  from  luxuriant,  greasy-looking, 
growths  on  glycerin- potato.  After  being  sub-cultivated  a  few  times  on  glycerin- 
potato  the  tubercle  bacillus  is  sown  in  cylindrical  flasks  with  a  flat  base  half  filled 
with  a  1  per  cent,  peptone  beef  broth  containing  6  per  cent,  glycerin.  The  cultures 
are  incubated  at  38°-39°  C.  and  shaken  daily.  It  is  necessary  to  sub-cultivate 
several  times  in  order  to  get  a  copious  growth. 

The  cultures  should  be  used  when  about  10  days  old :  later  they  are  thick  and 
too  rich  in  bacilli  and  are  only  partially  agglutinated  by  the  specific  serum. 

Cultures  sown  in  this  way  are  ^distinctly  cloudy  after  a  few  days,  and  when  shaken 
have  a  watered  silk  appearance  ;  the  cloudiness  subsequently  increases  and  becomes 
uniform,  and  after  2  or  3  weeks  the  growth  is  more  or  less  milky.  A  drop  of  the 
culture  examined  unstained  shows  small,  isolated  rods  occasionally  slightly  motile. 
The  bacilli  stain  by  Ziehl's  method  (except  in  rare  cases  recorded  by  Arloing  and  by 
Ferran).  On  inoculation  into  animals  they  behave  like  tubercle  bacilli  of  a  very 
low  degree  of  virulence. 

Ten-day-old  cultures  suitable  for  agglutination  can  be  kept  for  about  a  fortnight 
in  the  ice-chest  or  by  adding  a  little  formalin  (3-4  per  cent.)  to  them ;  the  aggmtina- 
bility  diminishes  after  a  few  weeks. 

To  obtain  and  to  keep  an  agglutinable  culture  more  easily  Arloing  and  Courmont 
now  advise  the  use  of  old  cultures  which  must  be  diluted  as  required.  The  culture 
is  left  in  the  incubator  for  30  or  40  days  and  can  be  used  at  once  or  kept  in  the  ice- 
chest  for  about  1  month.  When  required  for  use  the  culture  is  diluted  with  sterile 
normal  saline  solution  until  it  gives  a  milky,  slightly  opaque  fluid  like  a  solution  of 
glycogen  (about  1  in  50). 

When  a  culture  is  used  for  purposes  of  serum  diagnosis  a  control  should  always 
be  done  with  a  standard  serum  of  which  the  agglutinating  property  is  known  before- 
hand (the  serum  of  an  experimentally  infected  animal,  or  a  tuberculous  pleural 
exudate,  for  example). 

Homogeneous  cultures  are  agglutinated  by  the  serum  of  animals  which 
have  been  inoculated  with  tuberculin  or  living  bacilli  and  by  the  serum  of 
most  tuberculous  human  subjects  (66-90  per  cent.)  when  the  serum  is  diluted 
from  1  in  5  to  1  in  50.  The  degree  of  agglutinating  power  in  serums  from 
the  same  animal  species  appears  generally  speaking  to  bear  no  relation  to 


336  THE  TUBERCLE   BACILLUS 

the  severity  of  the  lesions  or  the  virulence  of  the  infecting  organism.  Serums 
from  cases  of  miliary  tuberculosis  often  fail  to  produce  the  reaction.  Tuber- 
culous serous  exudates  (peritonitis,  pleurisy)  in  most  cases  have  the 
property  of  agglutinating  the  tubercle  bacillus. 

The  technique  of  agglutination  is  as  follows  : 

Take  four  small  sterile  test-tubes  5-6  cm.  long.  A  little  of  the  pure  culture 
is  put  into  the  first  and  acts  as  a  control :  into  the  other  three  a  mixture  of 
serum  and  culture  is  introduced  in  different  proportions  :  thus,  into  the 
second  1  drop  of  serum  and  5  drops  of  culture,  into  the  third  1  of  serum  and 
10  of  culture  and  into  the  fourth  1  of  serum  and  15  of  culture.  After  shaking, 
the  tubes  are  placed  at  an  angle  of  45°  and  kept  at  laboratory  temperature. 
Agglutination  only  appears  after  the  mixtures  have  been  put  up  a  few  hours. 
The  tubes  should  be  examined  several  times  during  the  next  24  hours  and 
against  a  black  ground.  When  the  reaction  is  complete  the  fluid  is  clear 
and  there  is  a  slight  deposit  at  the  bottom  of  the  tube.  A  complete  reaction 
giving  this  appearance  is  alone  of  diagnostic  value.  The  diagnosis  should 
always  be  completed  by  microscopical  examination  of  the  clumps  (between  a 
slide  and  cover-glass  and  unstained). 

The  power  of  agglutination  possessed  by  the  serum  of  persons  suffering  from 
tuberculosis  is  of  very  limited  diagnostic  value  for  the  following  reasons  : 

1.  Homogeneous  cultures  are  often  agglutinated  by  the  serum  of  healthy  persons — 
26  times  out  of  100  examined  (Arloing  and  Courmont) :  50  per  cent,  of  cases  according 
to  German  statistics. 

2.  The  serum  of  persons  suffering  from  febrile  diseases  other  than  tuberculosis 
(enteric  fever,  puerperal  fever,  pneumonia,  etc.)  nearly  always  agglutinates  homo- 
geneous cultures  of  the  tubercle  bacillus. 

3.  The  serum  of  tuberculous  subjects  does  not  always  agglutinate  homogeneous 
cultures  of  the  infecting  organism.     A  negative  reaction  is  especially  frequent  in 
cases  of  advanced  phthisis,  miliary  tuberculosis,  and  "  galloping  "   consumption. 
The  reaction  is  often  absent  in  the  very  early  stages  of  the  disease,  and  is  practically 
never  obtained  in  cases  of  lupus.     Bezan9on,   Griffon  and  Philibert  obtained  a 
positive  reaction  in  about  83  per  cent,  of  cases  of  undoubted  tuberculosis  :    Ivanova 
observed  agglutination  in  66  per  cent,  of  cases  of  tuberculosis  and  in  60  per  cent, 
of  non-tuberculous  persons. 

4.  The  method  is  very  delicate  in  practice  and  the  most  minute  precautions  must 
be  taken  in  the  preparation  of  the  homogeneous  cultures. 

(ii)  Vasilescu  describes  a  medium  which  he  says  gives  homogeneous  cultures 
more  easily  than  Arloing 's  method. 

The  medium  has  the  following  composition : 

Clear  sterile  calf  serum,      -  25  c.c. 

Distilled  water,  75     ,, 

Neutral  glycerin,       -  3     „ 

Heat  the  mixture  to  100°  C.  in  a  water  bath,  distribute  into  tubes  of  2  cm.  diameter 
in  quantities  sufficient  to  give  a  column  3  cm.  high  in  each  tube.  Sterilize  at  120°  C. 
for  15  minutes  (the  medium  does  not  coagulate).  Sow  with  a  bacillus  of  the  human 
type.  Incubate  at  37°  C.  and  shake  the  tubes  every  day.  After  two  sub-cultures 
in  this  medium  the  growth  is  homogeneous. 

(iii)  Hawthorn  suggests  a  simplification  of  Arloing 's  technique.  He  sub- 
cultures an  homogeneous  culture  in  glycerin-broth  in  2  per  cent,  peptone 
saline  solution.  By  this  means  an  homogeneous  culture  consisting  of  motile 
bacilli  is  obtained  in  48  hours  without  shaking  the  flask.  These  cultures  are 
more  sensitive  for  the  agglutination  reaction  than  Arloing's. 

Vincent  repeated  Hawthorn's  experiments  and  always  found  that  the 
cultures  in  the  peptone  saline  medium  were  agglutinated  even  when  the 
serum  of  non-tuberculous  persons  was  used  (in  dilutions  of  1  in  30  and  1  in 


AGGLUTINATION— IMMUNE   BODIES  337 


40).     The  method  therefore  is  not  available  for  the  agglutination  reaction 
in  tuberculosis. 

(iv)  For  the  agglutination-reaction  German  authors  prefer  an  emulsion  of 
tubercle  bacilli  ground  up  in  a  mortar.  Von  Behring  uses  an  emulsion  of 
bacilli  triturated  in  an  agate  mortar  and  afterwards  desiccated.  Koch 
recommends  the  use  of  the  powdered  bodies  of  bacilli  obtained  in  the  pre- 
paration of  tuberculin  TK,  dried  and  emulsified  in  a  1  per  cent,  solution  of 
sodium  chloride.  In  bacillary  extracts,  obtained  by  triturating  tubercle 
bacilli  as  described  in  the  preparation  of  tuberculin  TK,  and  freed  from 
bacilli  by  centrifugation,  agglutinating  serums  produce  agglutination  in  dilu- 
tions of  1  in  10  to  1  in  50.  This  method  is  recommended  by  Koch  and 
Romberg. 

Kceppen  uses  an  emulsion  obtained  by  saponification  which  has  the 
advantage  of  keeping  well  and  of  being  of  such  a  degree  of  concentration 
that,  after  agglutination,  the  fluid  becomes  quite  clear. 

To  prepare  the  emulsion  described  by  Kceppen  filter  a  glycerin-broth  culture 
through  filter  paper  :  wash  the  bacilli  with  normal  saline  solution  and  dry,  in  vacuo, 
at  the  ordinary  temperature.  To  1  gram  of  dried  bacilli  add  3  c.c.  of  a  warm  aqueous 
solution  of  potash  (33  per  cent.) :  leave  to  stand  for  a  few  hours,  then  rub  up  the 
mixture  and  place  the  emulsion  in  the  warm  incubator  (37°  C.)  for  several  hours  ; 
finally  heat  in  a  water  bath  for  15  minutes  at  100°  C.  The  emulsion  is  now  of  a 
thick  consistency  and  is  again  heated,  the  water  which  evaporates  being  replaced 
by  alcohol  drop  by  drop.  Soaps  are  formed  which  are  dissolved  in  100  c.c.  of  warm 
distilled  water.  For  the  purposes  of  the  agglutination  reaction  1*5  c.c.  of  the  milky 
emulsion  is  mixed  with  50-100  c.c.  of  normal  saline  solution. 

(v)  Wright  and  Douglas  heat  a  glycerin-broth  culture  of  the  tubercle 
bacillus  to  60°  C.  for  1  hour,  filter  it  through  paper,  grind  it  up  in  a  mortar, 
and  emulsify  in  water  containing  0*1  per  cent,  of  sodium  chloride  and  0'5 
per  cent,  carbolic  acid.  They  then  centrifuge  the  emulsion  to  remove  any 
bacterial  masses  which  have  not  been  resolved  into  their  elements.  This 
liquid  is  only  agglutinated  by  normal  human  serum  in  dilutions  of  1  in  2  to 
1  in  4  while  tuberculous  serums  agglutinate  it  in  dilutions  of  1  in  10  to  1  in  50. 

6.  Immune  bodies  (Sensibilisatrices). 

The  presence  of  immune  bodies  is  very  inconstant  in  the  serum  of  persons 
suffering  from  tuberculosis.  The  method  of  complement  fixation  is,  there- 
fore, not  applicable  to  the  diagnosis  of  tuberculosis  (Widal  and  Le  Sourd, 
Camus  and  Pagniez,  Wassermann  and  others). 


SECTION  IV.— THE  DETECTION   OF  THE  TUBERCLE  BACILLUS. 

The  methods  employed  for  detecting  the  tubercle  bacillus  vary  in  detail 
according  to  the  nature  of  the  material  to  be  examined  but  in  every  case 
three  methods  of  investigation  are  available. 

1.  Microscopical  examination. — Fluids,  tissues  or  other  material  suspected 
to  contain  the  bacillus  must  be  stained  by  Ziehl's  or  Ehrlich's  method.  The 
tubercle  bacillus  is  the  only  organism  which  will  resist  the  decolourization 
used  in  these  methods.  As  a  matter  of  fact  two  other  [parasitic  human] 
organisms  might  be  mistaken  for  the  tubercle  bacillus,  namely,  the  bacillus 
of  leprosy  and  the  smegma  bacillus.1 

1  The  bacillus  of  verruga  can  be  left  out  of  account.  The  disease  has  so  far  been  very 
little  studied  and  is  unknown  in  these  climates. 


338  THE   TUBERCLE   BACILLUS 

The  methods  of  differentiating  the  former  are  considered  elsewhere  (Chap. 
XIX.).  In  the  case  of  the  smegma  bacillus  there  is  hardly  any  fear  of 
making  a  mistake  if  it  be  remembered  (i)  where  this  organism  is  found  and 
(ii)  that  though  it  resembles  the  tubercle  bacillus  in  resisting  the  decolourizing 
action  of  mineral  acids,  it  differs  from  it  in  being  rapidly  decolourized  by 
alcohol  (probably  because  alcohol  dissolves  the  fatty  matter  which  impregnates 
it).  In  short,  if  Ziehl-Neelsen's  method  be  carried  out  in  the  manner  described 
above  no  confusion  is  likely  to  arise  (p.  307).  To  avoid  all  possible  chance  of 
mistake  in  difficult  cases,  Bezan9on  and  Philibert  advise  staining  in  the  warm 
for  10  minutes,  decolourizing  in  33  per  cent,  nitric  acid  for  2  minutes  and  in 
absolute  alcohol  for  5  minutes. 

It  must  not  be  forgotten  that  saprophytic  acid-fast  bacilli  are  found  in  the 
ambient  media,  and  in  milk,  butter,  etc.  (p.  347). 

These  organisms  might  conceivably  be  a  serious  source  of  error  in  the 
detection  of  the  tubercle  bacillus.  But  they  are  often  only  feebly  resistant 
to  acid  and  are  frequently  decolourized  by  absolute  alcohol  alone.  If  there 
be  any  doubt  the  difficulty  can  be  cleared  up  by  inoculation. 

Films  are  prepared  in  the  ordinary  way.  Tissues  should  be  hardened  in 
alcohol  or  acid  perchloride.  [Eastwood  (for  the  English  Commission)  hardened 
tissues  in  10  per  cent,  formalin  for  a  few  days  then  washed  well  in  water  and 
transferred  to  Muller's  fluid.] 

2.  Inoculations. — In  those  cases,  which,  in  practice,  are  far  from  infrequent, 
in  which  microscopical  examination  fails  to  reveal  the  presence  of  the  tubercle 
bacillus  resort  must  be  had  to  animal  inoculation.     Guinea-pigs  being  the 
most  susceptible  animals  are  always  used  for  the  purpose.     When  the  material 
is  free  from  other  organisms,  as,  for  instance,  in  the  case  of  caseo-pus,  pleural 
fluid,  etc.  it  may  be  inoculated  into  the  peritoneal  cavity  but  in  other  cases 
(sputum,  pus  from  a  fistula,  etc.)  it  is  best  to  inoculate  it  beneath  the  skin 
otherwise  there  is  the  risk  of  setting  up  a  septic  peritonitis  which  will  kill  the 
animal  before  the  tubercle  bacillus  has  had  time  to  produce  its  characteristic 
lesions.     [Our    experience    would    lead    us    invariably   to   inoculate    intra- 
peritoneally  when  the  material  contains  other  organisms.     It  would  seem 
that  the  peritoneal  fluid  possesses  a  quality  lacking  in  the  sub-cutaneous 
tissues  of  destroying  putrefactive  and  other  organisms.] 

For  purposes  of  rapid  diagnosis,  Nattan-Larrier  and  Griffon  advise  the  inocula- 
tion of  the  suspected  material  into  the  mammary  gland  of  a  guinea-pig  during  the 
period  of  lactation.  The  bacilli  multiply  in  the  gland  and  after  a  week  or  a  fortnight 
they  can  be  detected  in  the  milk  by  staining  the  latter  by  Ziehl-Neelsen's  method. 

Osman  Nouri  has  drawn  attention  to  the  advantages  of  inoculating  an  animal  by 
rubbing  the  material  into  the  skin  after  shaving  it  (p.  299). 

Bloch  advises  that  the  suspected  material  should  be  inoculated  sub-cutaneously 
into  the  inguinal  region  of  a  guinea-pig  and  that  the  inguinal  glands  should  then  be 
squeezed  and  manipulated  between  the  fingers  to  bruise  them  and  so  render  them 
more  susceptible  to  infection.  Under  these  conditions,  if  the  material  (urine,  etc.) 
contained  the  tubercle  bacillus  the  glands  will  be  found  enlarged,  inflamed  and  even 
suppurating  when  the  animal  is  killed  9  days  after  inoculation.  The  method  is  not 
reliable ;  tubercle  bacilli  cannot  be  found  on  microscopical  examination  when  the 
animal  is  killed  9  days  after  inoculation ;  moreover  under  the  conditions  of  the 
experiment,  acid-fast  bacilli,  Staphylococci,  etc.  may  equally  with  the  tubercle 
bacillus  cause  swelling  and  suppuration  of  the  glands. 

3.  Cultures. — Cultures  are  very  rarely  used  as  a  means  of  detecting  the 
tubercle  bacillus  in  a  pathological  product.     To  obtain  cultures  not  only 
must  the  material  be  rich  in  tubercle  bacilli  [and  free  from  contaminating 
organisms]  but  there  must  be  a  good  deal  of  it.     Cultures,  however,  are 
particularly  successful  in  the  case  of  sputum. 


DETECTION   OF  THE   BACILLUS  339 

A.  Sputum. 

1.  Microscopical  examination. — The  search  for  tubercle  bacilli  in  sputum 
is  easy  when  the  latter  is  purulent  and  the  bacilli  are  present  in  large  numbers. 
It  is  much  more  difficult  and  often  unsuccessful  when  the  sputum  is  scanty 
and  mucous  in  character  and  derived  from  a  recent  lesion  or  again  when  the 
sputum  consists  almost  entirely  of  blood.  As  a  rule,  the  sputum  coughed  up 
by  the  patient  in  the  early  morning  gives  the  most  satisfactory  results. 

In  the  case  of  nummular  sputum  it  is  only  necessary  to  pick  up  a  small 
fragment  from  the  centre  of  a  purulent  mass  with  a  loop  and  spread  it  on  a 
cover-glass  [or  slide]  in  the  ordinary  way.  The  yellowish  lumps  found  in 
tuberculous  sputum  are  very  rich  in  bacilli  and  should  therefore  be  selected ; 
similarly,  in  mucous  sputum,  the  solid  particles  suspended  in  the  more  fluid 
portion  should,  as  far  as  possible,  provide  the  material  for  examination. 

It  is  very  difficult  to  find  the  tubercle  bacillus  in  the  blood  coughed  up 
during  an  attack  of  haemoptysis  ;  it  can  be  more  readily  found  in  the  con- 
solidated sputum  streaked  with  blood  which  is  expectorated  in  the  days 
following  the  haemorrhage. 

Bacilli  can  seldom  be  detected  in  the  sputum  of  persons  suffering  from 
miliary  tuberculosis;  [it  would  seem  that]  the  bacilli  only  pass  into  the 
sputum  when  purulent  lesions  are  breaking  down. 

The  tubercle  bacillus  is  present  also  in  the  expectoration  of  tuberculous 
cattle  (Riddoch)  and  a  good  method  of  diagnosing  the  disease  in  cattle  is  to 
collect  some  of  the  muco-purulent  material  from  the  partitions  in  the  sheds- 
and  to  examine  it  for  the  bacillus  after  staining  in  the  ordinary  way. 

Homogenization. — When  the  bacilli  are  likely  to  be  few  in  number  the 
sputum  must  be  specially  treated  before  they  can  be  detected  :  the  sputum 
must  be  liquefied  and  made  homogeneous,  and  then  be  left  to  deposit  or  else 
be  centrifuged.  Under  these  conditions  the  deposit  contains  in  a  small 
volume  all  the  bacilli  which  were  in  the  viscous  mass.  It  is  then  a  simple 
matter  to  stain  and  detect  them. 

1.  Biedert's  method. — To  15-20  c.c.  of  sputum  add  30-40  c.c.  of  water  and  a  few 
drops  (6-15)  of  soda — the  thicker  and  more  viscous  the  sputum  the  larger  must  be 
the  quantity  of  soda  added.     Boil  the  mixture  in  a  porcelain  dish  until  it  is  quite 
homogeneous,  then  dilute  with  one  or  two  volumes  of  water  and  boil  again  for  a 
minute  or  two.     Put  the  mixture  aside  for  48  hours,  then  pour  off  the  supernatant 
fluid  and  prepare  films  with  the  deposit.     Or,  after  boiling,  the  mixture  may  be 
centrifuged  and  the  deposit  used  for  making  films. 

2.  Ilkewitsch's  method. — Mix  0'5  c.c.  of  sputum  with  20  c.c.  of  distilled  water 
and  about  10  drops  of  a  3  per  cent,  solution  of  caustic  potash.     Heat  the  mixture 
in  a  porcelain  dish  but  without  letting  it  boil,  stirring  all  the  time  until  the  mixture 
is  homogeneous.     Add  a  little  casein  and  a  drop  or  two  of  a  3  per  cent,  solution  of 
caustic  potash  and  continue  the  heating  until  the  mixture  has  a  milky  appearance, 
then  pour  into  centrifuge  tubes,  add  a  few  drops  of  acetic  acid  until  coagulation  is 
just  beginning,  centrifuge  for  a  few  minutes  and  use  the  deposit  for  making  films. 

3.  Ellennann  and  Erlandsen's  method. — Mix  10-15  c.c.  of  sputum  with  one-half 
its  volume  of  a  0*6  per  cent,  solution  of  sodium  carbonate  in  a  small  flask  and  place 
in  the  incubator  at  37°  C.  for  24  hours.     Then  decant  the  supernatant  fluid  and 
centrifuge  the  remainder.     To  the  deposit  add  4  times  its  volume  of  0'25  per  cent, 
soda  solution,  mix,  centrifuge  again  and  use  the  deposit  for  preparing  films. 

According  to  Ellermann  and  Erlandsen,  and  Bertarelli,  this  method  is  better  than 
any  other  which  has  been  described. 

4.  Abba's  method. — Place  5-10  c.c.  of  sputum  in  a  cylindrical  vessel,  add  15-30  c.c. 
of  a  perchloride  solution  (perchloride  of  mercury,  2  grams  ;    salt,  10  grams  ;    dis- 
tilled water,  1000  c.c.) ;   mix  thoroughly,  centrifuge  15  c.c.  of  the  mixture  and  make 
films  with  the  deposit. 

5.  Sprengler's  method. — Mix  10  c.c.  of  sputum  with  10  c.c.  of  warm  water  and  a 


340  THE  TUBERCLE  BACILLUS 

drop  of  normal  soda  solution,  add  0 '25-0 '50  gram  of  pancreatin  and  incubate  at 
37°  C.  for  2  or  3  hours.  Then  centrifuge  or  pour  into  a  conical  glass  vessel  with  a 
crystal  of  thymol  added  to  hinder  putrefaction  and  leave  to  stand  for  12  or  14  hours  : 
decant  the  supernatant  liquid  and  prepare  films  with  the  deposit. 

6.  Jousset's  method. — Digest  the  sputum  for  2  or  3  hours  in  the  warm  incubator 
(38°  C.)  with  10-30  c.c.  of  artificial  gastric  juice  made  as  follows : 

Pepsin,    -  2  grams. 

Pure  glycerin.         1  __    in 

Hydrochloric  acid,}'  '     aa'  10  C'C' 

Sodium  fluoride,        -          -  3  grams. 

Distilled  water,  Q.S.  to  1000  c.c. 

When  completely  peptonized,  centrifuge  and  examine  the  deposit  for  tubercle 
bacilli  (p.  342). 

Films  made  from  sputum  or  from  the  deposit  after  solution  of  the  sputum 
are  stained  by  one  or  other  of  the  methods  already  described,  Ziehl-Neelsen's 
method  being  the  most  satisfactory. 

2.  Inoculations. — In  a  doubtful  case  of  pulmonary  phthisis  when  no  bacilli 
can  be  found  in  the  sputum  on  microscopical  examination,  animal  inoculation 
must  be  resorted  to  for  purposes  of  diagnosis.     The  sputum  must  be  collected 
as  free  from  other  organisms  as  possible  (p.  192)  and  an  emulsion  of  it,  pre- 
pared by  rubbing  up  in  sterile  water,  inoculated  beneath  the  skin  of  a  guinea- 
pig.     Should  the  sputum  contain  tubercle  bacilli  the  animal  soon  shows 
signs  of  infection  (p.  297).     On  account  of  the  risk  of  setting  up  a  fatal  septic 
peritonitis  sputum  should  never  be  inoculated  into  the  peritoneal  cavity  [but 
see  p.  338,  2.  Inoculations]. 

3.  Cultures. — It  is  very  rarely  that  cultures  are  sown  with  sputum  for 
purposes  of  diagnosis ;  indeed  for  a  long  time  it  was  thought  impossible  to 
obtain  a  pure  culture  of  the  bacillus  from  sputum.     Kitasato,  Pastor  and 
others  have,  however,  described  methods  by  which  it  may  be  effected. 

(a)  Kitasato' s  method. — After  the  mouth  has  been  washed  out  with  sterile  water, 
induce  the  patient  to  cough,  and  collect  the  sputum  in  a  sterile  glass  vessel.  Wash 
the  sputum  in  a  number  (10)  of  glass  vessels  containing  sterile  water  (p.  192)  then, 
adopting  the  ordinary  precautions  to  prevent  contamination,  lift  a  small  fragment 
from  the  centre  of  the  purulent  mass  and  spread  it  on  glycerin-serum.  Sow  a  large 
number  of  tubes.  Growth  appears  after  the  tubes  have  been  incubated  at  38°  C. 
for  about  10  or  12  days. 

(6)  Pastor's  method. — Collect  the  sputum  with  the  same  precautions  as  in  Kita- 
sato's  method.  Emulsify  in  a  little  sterile  water  and  filter  through  a  piece  of  sterile 
gauze.  Sow  a  tube  of  liquefied  gelatin  with  a  few  drops  of  the  filtrate,  pour  into  a 
Petri  dish  and  allow  it  to  set.  After  incubating  for  3  or  4  days  at  20°  C.  .any  con- 
taminating organisms  that  may  have  been  present  will  have  grown  giving  rise  to 
numerous  colonies.  With  a  sterile  scalpel  cut  out  those  portions  of  the  gelatin  on 
which  there  is  no  growth  and  transfer  them  to  tubes  of  glycerin-serum ;  if  a  number  of 
tubes  be  sown  a  pure  culture  of  the  tubercle  bacillus  will  be  obtained  on  some  of  them. 

(c)  Hesse's  method. — Hesse's  medium  (p.  317)  is  most  useful  for  isolating  the 
tubercle  bacillus  from  sputum.     The  sputum  is  washed  by  Kitasato's  method 
and  sown  on  the  agar  in  Petri  dishes.     After  incubating  at  37°  C.  for  about 
10  hours  the  number  of  tubercle  bacilli  in  the  fragments  of  sputum  will  be 
found  to  have  considerably  increased  and  colonies  will  be  visible  to  the 
naked  eye  at  about  the  sixth  day. 

(d)  Jockmann's  method. — Jockmann  uses  the  following  medium  which  is  similar 
to  Hesse's : 

Heyden's  albumose  (Nahrstoff  Heyden),       ....  5  grams. 

Salt,        -  5 

Neutral  glycerin,       -  30        ,, 

Normal  soda  solution,        -         -         -         -         -         -         -  5  c.c. 

Water, 1000     „ 


DETECTION   OF  THE   BACILLUS  341 

Sterilize.  To  20  c.c.  of  this  broth  add  10  c.c.  of  sputum,  mix  thoroughly  and 
incubate  at  37°  C.  After  incubating  for  24  hours  the  bacilli  will  have  increased  in 
number  and  films  can  be  prepared  with  the  deposit  and  stained  with  carbol-fuchsin. 
This  method  facilitates  the  detection  of  tubercle  bacilli  when  they  are  present  in 
the  sputum  in  small  numbers  only. 

(e)  Spengler's  method. — Tubercle  bacilli  are  more  resistant  to  the  action 
of  formalin  vapour  than  most  other  bacteria  and  it  is  upon  this  fact  that  the 
following  method  is  based.  Dworetzky  states  that  he  has  not  had  good 
results. 

Cover  the  bottom  of  a  Petri  dish  with  a  filter  paper,  spread  3  c.c.  of  sputum 
in  a  layer  not  more  than  2 '5  mm.  thick  and  sprinkle  with  pancreatin  to  facilitate 
digestion  of  the  mucus.  Line  the  lid  of  the  dish  with  filter  paper  soaked  in  a  few 
drops  of  commercial  formalin.  Incubate  the  dish  and  its  contents  at  20°-25°  C. 
After  incubating  for  2  hours  it  is  said  that  all  micro-organisms  other  than  the  tubercle 
bacillus  are  killed  so  that  if  tubes  of  glycerin-agar  be  now  sown  with  the  sputum 
a  pure  culture  of  the  tubercle  bacillus  will  be  obtained. 

[(/)  Griffith's  method. — A.  S.  Griffith  (for  the  English  Commission)  obtained 
cultures  direct  from  sputum  by  using  antiformin.1  The  method  he  employed 
at  first  was  that  recommended  by  Uhlenhuth ;  diluted  sputum  and  antiformin 
were  mixed  together  to  form  a  15  per  cent,  antiformin-sputum  mixture  and 
allowed  to  stand  for  3  hours ;  the  solution  was  then  centrifuged,  the  deposit 
washed  and  recentrifuged,  and  the  second  deposit  sown  on  to  culture 
media.  In  subsequent  experiments  however  he  found  that  a  10  per  cent, 
dilution  of  antiformin  allowed  to  act  for  20-30  minutes  gave  better  results 
(see  fig.  208  (a),  p.  317).] 

(g)  Spengler  recommends  another  method  based  on  the  resistance  of  the 
tubercle  bacillus  to  heat  and  only  applicable  to  nummular  sputum. 

Take  up  a  large  fragment  of  the  nummular  sputum  in  a  platinum  loop  and  hold  it 
near  a  flame  so  that  the  sputum  is  roasted  but  not  detached  from  the  loop.  Repeat 
the  process  three  or  four  times  then  sow  the  flamed  sputum  on  2  per  cent,  glycerin- 
serum  or  on  glycerin-agar.  Growth  appears  in  a  week  to  10  days.  Spengler  acknow- 
ledges that  this  requires  a  certain  amount  of  skill. 

In  a  case  of  advanced  pulmonary  tuberculosis  recorded  by  Bertarelli  the 
sputum  [appeared  to]  consist  solely  of  tubercle  bacilli  which  when  sown 
direct  on  to  glycerin-serum  and  blood-agar  readily  gave  a  pure  culture  of 
the  bacillus.  Such  cases  are  very  exceptional ;  and  undoubtedly  the  most 
certain  method  of  obtaining  a  pure  culture  from  sputum  is  to  inoculate  a 
guinea-pig  and  sow  cultures  from  the  lesions  which  develop  (p.  314). 

B.  Blood. 

The  tubercle  bacillus  rarely  passes  into  the  blood  of  persons  affected  with 
tuberculosis.  Lustig  and  others  have,  however,  succeeded  in  staining  the 
bacillus  in  films  prepared  with  blood  obtained  by  pricking  the  finger  or  the 
spleen  (in  miliary  tuberculosis).  The  bacillus  is  more  easily  demonstrated 
in  the  clots  formed  post  mortem  in  the  heart  and  blood  vessels.  The  carbol- 
fuchsin  method  should  be  employed  for  staining  the  preparations. 

Bezangon  and  Griffon,  and  Jousset  have  described  methods  designed  to  facili- 
tate the  detection  of  the  organism  in  the  blood.  The  results  of  the  methods  are, 
however,  vitiated  by  the  occurrence  in  the  surrounding  air  of  acid-fast  bacilli 
and  of  saprophytic  bacilli  which  acquire  acid-fast  properties  in  organic 
products.  To  secure  the  best  results  from  these  methods  they  ought  to  be 
carried  out  under  strictly  aseptic  conditions,  and  this  in  practice  is  difficult 

[l  Antiformin  is  a  mixture  of  sodium  hydroxide  and  Eau  de  Javelle.  It  has  the  power 
of  dissolving  albuminous  substances  and  of  killing  and  dissolving  all  bacteria  except 
those  which  like  the  tubercle  bacillus  possess  a  waxy  envelope.] 


342  THE   TUBERCLE   BACILLUS 

of  accomplishment.  Under  strictly  aseptic  conditions,  Bergeron  has  re- 
peatedly failed  to  detect  the  presence  of  tubercle  bacilli  in  blood  by  Jousset's 
method. 

(a)  Bezangon  and  Griffon's  method. — To  5  c.c.  of  blood  add  5  c.c.  of  distilled  water 
and  5  drops  of  soda  and  triturate  the  mixture  in  a  mortar  until  completely  dissolved. 
Then  add  20  c.c.  of  water  and  boil  in  a  porcelain  dish  for  5  minutes.  Centrifuge 
the  product  for  10  minutes,  prepare  films  and  stain  with  carbol-fuchsin. 

(&)  Jousset's  method  (Inoscopy). — To  30  c.c.  of  blood  add  100  c.c.  of  distilled 
water.  Digest  the  clot  with  artificial  gastric  juice  (p.  340)  for  2  or  3  hours  in  the 
warm  incubator  (38°  C.).  Centrifuge  the  product,  stain  the  deposit  with  carbol- 
fuchsin,  and  examine  it  for  tubercle  bacilli. 

(c)  Nattan-Larrier  and  Bergeron's  method.— In  this  method  the  blood  is 
received  direct  from  the  vein  into  twenty  times  its  volume  of  sterile  distilled 
water ;  the  blood  hsemolyses ;  the  haemolyzed  mixture  is  centrifuged  and  the 
deposit  examined  for  bacilli. 

(d)  Blood  may  be  collected  in  sodium  citrate  solution  to  prevent  it  clotting, 
centrifuged  and  the  deposit  examined.     Lesieur  utilizes  the  anti-coagulating 
property  of  the  digestive  juices  of  leeches.     A  leech  is  put  on  the  patient  and 
when  gorged  with  blood  it  is  pressed  and  the  product  centrifuged. 

C.  Pus. 

In  pus  from  a  tuberculous  lesion  the  bacilli  are  present  in  small  numbers 
only  so  that  search  for  the  organism  in  films  often  has  a  negative  result.  One 
or  other  of  the  methods  of  homogenization  described  above  when  dealing 
with  sputum  may  with  advantage  be  adopted,  though  it  is  always  pre- 
ferable to  inoculate  a  guinea-pig.  The  tubercle  bacillus  in  the  majority  of 
cases  occurs  in  pure  culture  in  tuberculous  pus  but  in  other  cases  it  may 
be  associated  with  the  ordinary  pyogenic  organisms  and  particularly  with 
staphylococci. 

D.  Exudates. 

In  the  sero-fibrinous  exudates  which  occur  in  pleurisy,  peritonitis,  peri- 
carditis, etc.,  direct  examination  for  the  tubercle  bacillus  by  microscopical 
examination  always  gives  negative  results.1 

Jousset  appears  to  have  obtained  remarkable  results  by  applying  the 
method  of  inoscopy  to  the  detection  of  the  tubercle  bacillus ;  he  found  the 
bacillus  in  all  sero-fibrinous  exudates.  Unfortunately  this  method  involves  risk 
of  error  by  reason  of  the  presence  of  acid-fast  bacilli  in  the  surrounding  air 
(p.  341)  and  the  bacilli  stained  by  Jousset  were,  at  least  in  most  cases,  evidently 
not  the  tubercle  bacillus.  Jousset  himself  noted  their  abnormal  forms  and 
the  ease  with  which  they  were  decolourized  by  too  long  immersion  in  acid 
(p.  345).  Moreover,  the  method  of  inoscopy  usually  gives  negative  results 
when  it  is  applied  under  strictly  aseptic  conditions  (Bergeron). 

Jousset's  technique. — If  the  liquid  be  spontaneously  coagulable  it  is  allowed  to 
clot  and  the  clot  treated  as  described  above  in  the  case  of  blood.  When  the  liquid 
does  not  coagulate  spontaneously  (cerebro-spinal  fluid,  for  example)  some  horse- 
blood  plasma  is  added  to  form  a  clot  and  this  is  then  treated  in  the  ordinary  way. 
Horse  plasma  is  obtained  by  mixing  equal  volumes  of  horse- blood  and  10  per  cent, 
solution  of  sodium  chloride,  centrifuging  and  collecting  the  supernatant  fluid. 

Satisfactory  results  may  be  obtained  by  sowing  the  exudate  on  blood- 

P  Though  this  statement  is  true  in  the  majority  of  cases  its  application  is  not  so  universal 
as  the  author's  experience  would  lead  him  to  think.  Microscopical  examination  of  fluid 
from  cases  of  tuberculous  pleurisy  may  show  the  presence  of  tubercle  bacilli  and  occa- 
sionally in  extraordinarily  large  numbers.] 


DETECTION   OF  THE   BACILLUS  343 

agar.  Bezancon  and  Griffon  obtained  cultures  in  12-15  days  from  ten  cases 
of  tuberculous  meningitis  by  sowing  the  cerebro-spinal  fluid.  And  the 
same  authors  obtained  cultures  of  the  tubercle  bacillus  in  two  cases  of  sero- 
fibrinous  pleurisy. 

The  classical  method  is  to  inoculate  a  guinea-pig  with  the  suspected  fluid. 
But  in  this  connexion  it  must  be  borne  in  mind  that  inoculation  of  tuberculous 
pleural  fluid  gives  negative  results  in  three-quarters  of  the  cases.  [This 
is  probably  because  the  fluid  is  actually  free  from  tubercle  bacilli  since  "an 
extremely  small  number  of  bacilli  is  able  to  induce  a  progressive  tuberculosis 
in  the  guinea-pig  "  (A.  S.  Griffith,  for  the  English  Commission).]  Inoculation 
is  best  done  into  the  peritoneum  with  a  large  quantity  (10-15  c.c.)  of  the  fluid, 
which  must,  of  course,  be  collected  aseptically.  To  ascertain  the  degree  of 
virulence  of  the  bacillus  a  rabbit  should  be  inoculated  at  the  same  time,  for 
a,  bacillus  which  will  infect  a  guinea-pig  often  produces  no  lesion  in  a  rabbit 
(Arloing).  [Rabbits  inoculated  with  very  small  doses  of  the  human  tubercle 
bacillus  frequently  show  no  tuberculous  lesions  when  killed  (English  Com 
mission).] 

Debove  and  Renault's  method. — Debove  and  Renault  have  devised  a  very  ingenious 
method  for  deciding  the  nature  of  a  suspected  tuberculous  exudate.  They  showed 
that  tuberculous  exudates  contain  tuberculin.  The  inoculation  of  a  small  quantity 
of  a  pleural  or  pericardial  exudate  into  a  tuberculous  guinea-pig  gives  the  charac- 
teristic tuberculin  reaction  (p.  324). 

E.  Granulomata. 

In  the  majority  of  cases  microscopical  examination  fails  to  reveal  the 
presence  of  the  tubercle  bacillus.  A  small  piece  of  the  growth  should  in  this 
event  be  inoculated  beneath  the  skin  of  a  guinea-pig. 

F.  Nasal  cavities. 

Strauss  has  shown  that  tubercle  bacilli  are  frequently  found  (once  out  of 
three  times)  in  the  nasal  fossse  of  healthy  subjects  living  in  close  contact 
with  persons  suffering  from  phthisis.  The  following  paragraph  describes 
Strauss'  technique. 

Prepare  a  number  of  small  swabs  by  rolling  a  little  piece  of  absorbent  wool  round 
the  end  of  a  small  stick  of  wood  (10-15  cm.  long)  [or  stout  iron  wire]  and  sterilize 
them  in  wool-plugged  test  tubes  in  the  hot  air  sterilizer.  Pass  one  of  these  sterile 
swabs  into  the  nasal  cavity  and  by  rubbing  it  gently  over  the  mucous  membrane 
collect  the  dust  and  mucus  adhering  to  it.  Wash  the  swab  in  a  little  sterile  water. 
Repeat  the  operation  six  or  eight  times  in  each  case  and  wash  the  different  swabs 
in  the  same  water,  then  inoculate  the  emulsion  into  the  peritoneal  cavity  of  a  guinea- 
pig- 

G.  Urine. 

Microscopical  examination  for  tubercle  bacilli  of  the  urine  of  patients 
affected  with  tuberculosis  of  the  urinary  passages  often  gives  negative  results 
even  when  the  urine  has  been  centrifuged  and  the  deposit  used  for  examination. 
It  must  not  be  expected  that  large  numbers  of  bacilli  will  be  found  even  in 
the  most  favourable  cases :  should,  however,  a  large  number  of  acid-fast 
bacilli  be  found  on  microscopical  examination  of  a  urine  the  result  should  be 
regarded  with  suspicion  and  the  examination  done  again,  decolourizing  with 
alcohol  for  a  long  time.  This  would  be  a  typical  case  for  inoculation. 

In  cases  of  acute  tuberculosis  even  when  there  is  no  lesion  of  the  urinary 
passages  the  tubercle  bacillus  may  pass  into  the  urine  (Benda,  Weichselbaum, 
L.  Fournier  and  Beaufume). 


344  THE   TUBERCLE   BACILLUS 

Pour  the  urine  into  a  conical  glass  vessel  and  add  a  small  crystal  of  thymol 
or  camphor.  If  there  is  an  abundant  deposit  of  pus  homogenize  the  deposit 
and  centrifuge.  If  only  a  small  deposit  is  formed,  decant  the  supernatant 
liquid  and  prepare  films  with  the  deposit.  If  after  24  hours  there  is  only  a 
minimal  deposit,  decant  the  upper  part  of  the  liquid,  add  an  equal  volume  of 
95  per  cent,  alcohol  to  the  few  cubic  centimetres  of  liquid  remaining  in  the 
vessel,  mix  and  centrifuge. 

•     <v 
' 


FIG.  212.— Tubercle  bacilli  in  urine.    (Carbol-fuchsin  and  methylene  blue.) 
(Oc.  2,  obj.  Ath,  Zeiss.) 

When  a  urine  contains  only  a  few  cells  the  deposit  adheres  badly  to  the 
cover-glasses  and  is  liable  to  be  washed  off  in  the  staining  process.  This 
difficulty  is  especially  encountered  when  the  urine  yields  a  large  bulky  pre- 
cipitate of  crystals  of  urates  on  centrifuging.  To  overcome  this,  Trevithic 
recommends  washing  the  deposit  several  times  in  distilled  water  but  the 
method  does  not  seem  altogether  reliable.  The  author  prefers  to  heat  the 
urine  to  40-45°  C.,  centrifuge  and  wash  the  deposit  once  with  distilled  water 
at  45°  C.  When  the  deposit  is  very  small,  it  may  be  mixed  with  2  or  3  drops 
of  a  mixture  of  fresh  egg  albumin  and  distilled  water  (1-3)  which  fixes  the 
deposit  on  the  slide  better. 

Jousset's  method  has  been  utilized  for  the  detection  of  the  tubercle  bacillus  in 
urine.  Add  some  blood  plasma  to  the  urine,  digest  the  clot  which  forms  with  artificial 
gastric  juice  (p.  340)  and  centrifuge.  Examine  the  deposit  for  tubercle  bacilli.  It 
must  not  be  forgotten  that  this  method  more  than  any  other  is  liable  to  lead  to  error 
on  account  of  the  presence  of  other  acid-fast  bacilli — particularly  of  the  smegma 
bacillus  which  is  easily  mistaken  for  the  tubercle  bacillus. 

The  only  certain  method  of  detecting  the  tubercle  bacillus  in  urine  is  to 
inoculate  a  guinea-pig.  When  the  urine  can  be  collected  aseptically  and  is 
not  contaminated  either  with  the  colon  bacillus  or  other  pyogenic  organisms 
a  few  cubic  centimetres  may  be  inoculated  into  the  peritoneal  gavity  of  a 
guinea-pig.  In  the  contrary  case  the  urine  should  be  inoculated  sub-cutane- 
ously.  It  has  also  been  recommended  that  the  deposit  obtained  on  centri- 
fuging the  digested  clot  in  Jousset's  method  should  be  inoculated. 


H.  Excreta. 

[Acid-fast  bacilli  having  the  morphological  properties  and  staining  reactions 
of  the  tubercle  bacillus  can  often  be  seen  in  films  made  with  the  excreta  of 


I 


DETECTION  OF  THE   BACILLUS  345 

tuberculous  subjects  and  sometimes  in  very  large  numbers.  In  order  to 
determine  whether  these  bacilli  are  tubercle  bacilli  or  no  resort  must  always 
be  had  to  guinea-pig  inoculation.] 

I.  Milk. 

Tubercle  bacilli  occur  only  in  small  numbers  in  milk  and  the  chances  of 
finding  them  by  microscopical  examination  are  far  from  great.  [Moreover 
non-pathogenic  acid-fast  bacilli  (infra)  are  of  frequent  occurrence  in  cow's 
milk  and  no  reliance  can  be  placed  upon  microscopical  examination  for  the 
detection  of  tubercle  bacilli  in  milk.  ]  Several  methods  have  been  described 
for  detecting  the  bacillus  in  milk. 

(a)  Leave  the  fresh  milk  to  stand  for  24  hours  and  examine  the  deposit. 

(6)  Centrifuge  and  use  the  precipitate  for  making  films. 

(c)  Coagulate  200  c.c.  of  milk  with  a  little  powdered  citric  acid,  filter, 
dissolve  the  precipitate  on  the  filter  in  a  solution  of  sodium  phosphate,  pour 
the  liquid  into  a  large  test-tube,  add  a  few  cubic  centimetres  of  ether,  shake 
for  10  minutes  or  so,  decant  the  ether  with  the  fat  in  suspension,  centrifuge 
the  aqueous  fluid  and  examine  the  deposit. 

With  milk  as  with  urine  the  only  certain  method  of  ascertaining  whether 
a  given  specimen  contains  the  tubercle  bacillus  is  to  inoculate  a  few  cubic 
centimetres  collected  as  aseptically  as  possible  into  the  peritoneum  of  a  guinea- 
pig.  [Stand  the  milk  in  the  ice  chest  for  12  hours.  Pipette  off  some  of  the 
cream  into  one  sterile  centrifuge  tube  and  the  deposit  into  another.  Centri- 
fuge and  inoculate  3  c.c.  of  the  cream  from  the  first  tube  into  one  guinea-pig 
and  3  c.c.  of  the  deposit  from  the  second  into  another  guinea-pig.] 

[A.  S.  Griffith  and  others  have  shown  that  tubercle  bacilli  are  found  in  the 
milk  of  cows  suffering  from  tuberculosis  quite  independently  of  whether  there 
is  disease  of  the  udder  or  not.  The  bacilli  cannot  however  be  detected  on 
every  occasion  on  which  the  milk  is  tested.  F.  Griffith  suggests  in  explana- 
tion of  this  fact  "  that  the  quantity  of  milk  "  (50  c.c.)  "  inoculated  was  not 
sufficient  to  be  representative  rather  than  that  the  elimination  of  tubercle 
bacilli  was  irregular  since  in  several  of  the  animals  "  (inoculated  with  the 
milk)  "  the  slight  amount  of  disease  produced  showed  that  only  a  few  bacilli 
had  been  inoculated."  And  this  explanation  is  supported  by  the  fact  that 
if  a  number  of  guinea-pigs  say  eight  be  each  inoculated  with  the  same  quantity 
of  the  same  milk  often  not  more  than  one  animal  develops  tuberculosis.  ] 


THE   ACID-FAST,   OR   PARA-TUBERCLE,   BACILLI. 

Besides  the  leprosy  bacillus,  the  bacillus  of  verruga  and  the  smegma  bacillus 
there  is  a  number  of  bacilli  which  share  with  the  tubercle  bacillus  the  property  of 
resisting  the  decolourizing  action  of  acids.  These  organisms,  variously  described 
as  acido-phile,  acid-fast,  or  para-tubercle  bacilli,  have  been  described  by  Petri, 
Rabinowitsch,  Riibner,  Beck,  Obermiiller,  Coggi,  and  Moeller  as  occurring  in  milk, 
butter,  manure,  grass,  air,  and  so  on.  Moeller,  in  particular,  has  described  several 
species  of  these  organisms  (the  manure  bacillus  or  Misibazillus,  the  grass  bacillus  or 
Grasbazillus,  and  the  Timothy-grass  bacillus  or  Timothee  bazillus).  Similar  bacilli 
have  been  found  in  various  pathological  conditions  in  man  e.g.  in  gangrene  of  the 
lung  (Pappenheim,  Meyer,  Lydia  Rabinowitsch,  and  others)  in  conditions  of  the 
eye  simulating  tuberculosis  (Guisberg),  in  various  pulmonary  diseases  (Moeller, 
Flexner,  Ohlmacher,  and  others)  and  in  diseases  of  the  alimentary  canal  (Mironescu) 
etc. — The  ichthic  bacillus  described  by  Dubard  also  belongs  to  this  group. 

None  of  these  bacilli  which  morphologically  resemble  the  tubercle  bacillus  can 
be  distinguished  from  the  latter  in  microscopical  preparations  :  when  stained  they 
are  not  decolourized  by  acid  and  even  sometimes  not  by  alcohol.  According  to 


346  THE  PARA-TUBERCLE    BACILLI 

Borrel  their  morphological  characteristic  as  in  the  case  of  the  tubercle  bacillus  is 
to  remain  a  bright  red  colour  when  treated  in  the  following  manner 

1.  Stain  with  carbol-fuchsin  in  the  warm  for  5  or  10  minutes. 

2.  Treat  with  2  per  cent,  aniline  hydrochloride  for  1  or  2  minutes. 

3.  Decolourize  in  absolute  alcohol. 

4.  Differentiate  with  a  dilute  aqueous  solution  of  methylene  blue. 

They  are  distinguished  from  the  tubercle  bacillus,  (1)  by  the  ease  with  which  they 
can  be  grown  on  various  media  containing  no  glycerin  at  the  ordinary  temperature 
of  the  laboratory  (2)  by  their  cultural  characteristics  (luxuriant  and  generally 
greasy  and  creamy)  and  (3)  finally  and  especially,  by  the  fact  that  they  do  not  produce 
tuberculin.  Ramond  and  Ravaut,  Bataillon  and  Terre  have  described  an  ichthic 
tuberculin  similar  to  human  tuberculin  but  their  results  have  not  been  confirmed 
(p.  296). 

Some  of  the  members  of  this  group  are  pathogenic  to  animals,  particularly  guinea- 
pigs,  and  may  set  up  either  local  lesions  distinctly  tuberculous  in  appearance 
or  pseudo-miliary- tuberculoses  (Timothee  bazillus)  which  have  a  tendency  to 
suppurate. 

Finally,  several  authors  have  directed  attention  to  the  existence  of  pseudo-acid- 
fast  bacilli  (Bezangon  and  Philibert,  Bienstock,  and  others).  A  large  number  of 
saprophytic  bacilli  as  a  result  of  living  in  contact  with  fatty  substances  acquire, 
accidentally,  as  it  were,  acid-fast  properties  (e.g.  B.  smegmce]  which  are  lost  as  soon 
as  they  are  grown  on  ordinary  culture  media.  Other  saprophytic  bacilli  become 
acid-fast  when  grown  in  blood  or  sero-fibrinous  exudates  (Bezancon  and  Philibert). 
The  Bacillus  anthracis,  Bacillus  subtilis  (Bienstock),  Bacillus  entericce  febris  (Ramond 
and  Ravaut),  and  Bacillus  diphtherice  (Bezan9on  and  Philibert)  become  acid-fast 
when  grown  in  media  containing  butter.  But  all  these  pseudo-acid-fast  bacilli  are 
decolourized  by  prolonged  treatment  with  acid  and  especially  by  alcohol  (pp.  306  and 
342)  ;  and  moreover,  unlike  the  tubercle  bacillus,  they  can  be  stained  with  Unna's 
blue  (10  minutes). 

There  should  therefore  be  no  reason  for  confusing  the  tubercle  bacillus  with  the 
para-tubercle  bacilli. 

The  smegma  bacillus. 

Tavel,  Alvarez,  Matterstock  have  isolated  from  normal  smegma  a  bacillus  which 
resists  decolourization  by  acids.  This  is  the  bacillus  which  Lustgarten  described  as 
the  cause  of  syphilis.  It  has  not  been  grown  outside  the  body  and  is  decolourized 
by  acid-alcohol ;  it  should  not  therefore  be  difficult  to  differentiate  it  from  the 
tubercle  bacillus.  Houssell's  method  is  the  best  for  purposes  of  micro-chemical 
diagnosis.  The  technique  is  as  follows. 

Stain  the  film  in  the  warm  for  2  minutes  with  carbol-fuchsin.  Wash.  Treat 
for  10  minutes  with  acid-alcohol. 

Absolute  alcohol,       -         -  -         100  c.c. 

Pure  hydrochloric  acid,      -  3     „ 

Wash  again.     Stain  with  an  aqueous  solution  of  methylene  blue. 

Saturated  aqueous  solution  of  methylene  blue,     -  50  c.c. 

Distilled  water,  50     „ 

Wash.     Dry.     Mount.     The  smegma  bacillus  will  be  stained   blue,   the  tubercle 
bacillus,  red. 

The  bacillus  of  verruga  peruana. 

Verruga  is  a  disease  found  in  certain  valleys  of  the  Andes.  It  affects  man  and 
some  of  the  domestic  animals.  It  occurs  both  as  an  acute  and  chronic  disease  and 
leads  to  the  formation  of  granulomata  on  mucous  surfaces,  the  skin  and  the  viscera 
(Odriozola).  Carriou  inoculated  himself  with  the  blood  of  a  person  suffering  from 
the  chronic  form  of  the  disease  and  died  of  an  acute  infection.  A  dog,  inoculated 
by  Tamayo  with  1  c.c.  of  the  blood  of  a  person  suffering  from  verruga  in  an  acute 
form,  became  infected  with  a  typical  attack  of  the  disease  and  recovered.  According 
to  Ch.  Nicolle  and  Letulle,  the  cause  of  verruga  is  a  bacillus  morphologically  identical 
with  the  tubercle  bacillus  and  staining  by  the  Ziehl-Neelsen  method.  The  organism 
has  not  been  cultivated. 


THE   PSEUDO-TUBERCULOSES  347 


The  Pseudo-tuberculoses. 

In  addition  to  the  tubercle  bacillus  there  is  a  certain  number  of  other  pathogenic 
organisms  capable  of  producing  tubercles  in  the  tissues. 

The  bacillus  of  leprosy  and  of  glanders  both  lead  to  the  formation  of  true  tubercles. 
In  connexion  with  the  Dlscomyces  it  will  be  seen  that  some  of  the  members  of  that 
group  produce  pseudo-tuberculous  conditions  in  man  and  the  lower  animals. 

Finally  some  bacteria  give  rise  to  lesions  so  closely  simulating  those  produced  by 
the  tubercle  bacillus  that  they  may  be  mistaken  for  tuberculous  lesions.  These 
pseudo-tuberculous  lesions  may  be  classified  into  two  groups,  namely  the  zoogleic 
pseudo-tuberculoses  of  Malassez  and  Vignal,  Chantemesse,  and  others :  and  the 
bacillary  pseudo-tuberculoses  of  Charrin  and  Roger,  Dor,  Courmont,  and  others. 

The  descriptions  given  by  different  authors  do  not  at  all  coincide  :  perhaps  they 
relate  to  a  number  of  varieties  of  the  same  organism.  It  must  suffice  to  have 
recorded  the  existence  of  this  group  of  organisms  :  a  description  of  them  is  beyond 
the  scope  of  this  work. 

[The  disease  commonly  known  as  pseudo-tuberculosis  in  guinea-pigs  and  rabbits 
is  briefly  described  at  p.  160.] 


CHAPTEE  XIX. 
BACILLUS  LEPR^B. 

Introduction. 

Section  I. — Attempts  to  reproduce  the  disease  experimentally,  p.  348. 

Section  II. — Morphology,  p.  350. 

Section  III. — Serum -therapy,  p.  353. 

Section  IV.: — Detection  and  identification  of  the  leprosy  bacillus,  p.  354. 

LEPROSY,  the  cause  of  which  is  a  bacillus  discovered  by  Hansen,  is  a  con- 
tagious disease  peculiar  to  man  :  the  lower  animals  are  never  infected.  A 
disease  apparently  very  similar  to  human  leprosy  has  however  been  described 
as  occurring  in  the  rat  (Rabinowitsch  and  others)  ;  it  takes  the  form  of 
ulcers  on  the  skin  and  swelling  of  the  glands,  and  the  lesions  contain  very 
large  numbers  of  bacilli  similar  to  the  leprosy  bacillus.1 

[Though  there  is  no  definite  and  absolutely  conclusive  proof  of  the  setio- 
logical  role  of  the  organism  hitherto  commonly  known  as  the  bacillus  leprce 
there  can  be  no  reasonable  doubt  bat  that  the  parasite  is  the  cause  of  leprosy. 
Recent  investigations  however  would  seem  to  afford  amply  sufficient  ground 
for  believing  that  the  organism  is  not  a  true  bacterium  but  rather  an  hypo- 
mycete  belonging  to  the  genus  Discomyces  (Streptothrix).  This  being  so  it 
would  be  more  exact  to  supersede  its  present  designation  by  the  name  pro- 
posed by  Deycke  :  Streptothrix  leproides.  These  researches  are  also  of  interest 
in  that  they  afford  botanical  evidence  of  the  close  relationship  which  has  on 
other  evidence  been  known  for  long  enough  to  exist  between  the  parasites 
of  leprosy  and  tuberculosis.] 


SECTION  I.— ATTEMPTS  TO  REPRODUCE  THE  DISEASE 
EXPERIMENTALLY. 

Most  of  the  attempts  made  to  reproduce  the  disease  experimentally  by 
inoculating  the  bacillus  have  failed. 

In  man,  Arning  is  said  to  have  succeeded  in  inoculating  with  leprosy  the  condemned 
criminal  Keanu,  but  in  this  case  the  hypothesis  of  a  spontaneously  contracted 
infection  may  be  pleaded  :  the  same  objection  may  be  raised  against  two  or  three 
other  cases  in  which  leprosy  is  said  to  have  been  successfully  transmitted  by  inocula- 
tion. Against  these  experiments  of  doubtful  validity  must  be  placed  the  very 
numerous  unsuccessful  attempts  made  by  a  number  of  different  observers. 


[*  This  disease  is  endemic  in  England,  on  the  Continent,  in  Australia,  in  America,  and 
in  Japan.] 


EXPERIMENTAL  INOCULATION  349 

In  the  lower  animals,  with  the  probable  exception  of  the  monkey,  the  inocu- 
lation of  leprous  tissues  or  cultures  of  the  organism  produces  no  result  [but  vide 
infra]. 

In  the  lesions  found  by  Melcher  and  Orthmann,  and  Tedeschi,  after  the  inocula- 
tion of  a  rabbit  with  pieces  of  leprous  tissue,  the  presence  of  what  was  probably  the 
tubercle  bacillus  as  well  as  other  organisms  unconnected  with  leprosy  was  demon- 
strated. Thiroux,  in  Madagascar,  inoculated  four  rabbits  with  leprous  nodules  : 
all  four  animals  developed  typical  and  fatal  tuberculosis  ;  the  tissues  were  sown 
and  yielded  cultures  of  the  tubercle  bacillus.  Numerous  inoculation  experiments 
carried  out  on  monkeys  (Babes),  pigs  (Hilairet  and  Gaucher,  Widal),  dogs  (Neisser, 
Danisch),  rabbits  (Wesener),  and  cold-blooded  vertebrata  (Kobner),  and  more 
recently  the  experiments  of  Besnier  and  Leloir,  have  all  failed  to  give  rise  to  leprosy 
in  the  inoculated  animal. 

A  piece  of  leprous  tissue  inoculated  beneath  the  skin  of  an  animal  retains  its  normal 
appearance  for  a  long  time,  and  the  bacilli  contained  in  it  will  stain  even  after  the 
lapse  of  several  months,  but  they  never  undergo  any  multiplication.  The  tissue 
is  gradually  absorbed  by  leucocytes  which  congregate  around  it. 

C.  Nicolle  has  succeeded  in  inoculating  macacus  monkeys  with  leprosy. 

A  non-ulcerated  leprous  nodule  was  pounded  in  a  mortar  and  inoculated  into  the 
ear  of  a  bonnet  monkey  (Macacus  sinensis) :  sixty-two  days  later  leprous  nodules 
containing  quite  typical  leprosy  bacilli  were  found  to  have  developed.  Six  other 
macacus  monkeys  (Macacus  sinensis  and  Macacus  rhesus)  are  said  to  have  been 
successfully  inoculated  beneath  the  skin  :  by  repeating  the  inoculations  the  suscepti- 
bility of  the  monkey  is  increased  and  the  period  of  incubation  can  be  reduced  from 
two  months  to  a  fortnight  (fourth  inoculation).  The  lesions  resolved  spontaneously 
in  29-160  days.  For  purposes  of  inoculation,  material  rich  in  bacilli  from  untreated 
lesions  should  be  used. 

[Host  also  successfully  infected  a  monkey  by  repeatedly  inoculating  it  with 
an  organism  which  he  had  cultivated  from  a  case  of  leprosy.  The  monkey 
exhibited  all  the  clinical  features  of  tuberculous  leprosy  and  in  the  nodules 
acid-fast  bacilli  were  found  situated  as  in  leprosy. 

[Host  failed  in  his  attempts  to  infect  guinea-pigs,  white  rats  and  rabbits 
either  by  inoculation  (sub-cutaneous  and  intra-peritoneal)  or  by  feeding. 

[Bayon  inoculated  rats  and  mice  with  an  acid-resisting  diphtheroid  bacillus 
which  he  had  cultivated  from  cases  of  human  leprosy  and  found  that  the 
organism  when  recovered  from  the  organs  of  these  animals  had  acid-fast 
properties.  This  acid-fast  organism  when  inoculated  into  a  further  series  of 
rats  and  mice  caused  "  the  identical  changes  of  genuine  spontaneous  rat 
leprosy  and  very  striking  analoga  of  the  glands  and  organs  of  human 
cases." 

[Williams  with  his  pleomorphic  streptothrix  (vide  infra)  produced  in 
guinea-pigs  by  sub-cutaneous  inoculation  a  lesion  somewhat  resembling 
leprosy  with  large  numbers  of  cocco-bacilli  in  the  cells  of  the  connective 
tissue. 

[Duval  used  cultures  2-3  days  old  on  glycerin-blood-agar.  By  inoculating 
monkeys  (Macacus  rhesus)  sub-cutaneously  two  or  three  times  at  intervals 
he  was  able  to  produce  lesions  resembling  those  of  leprosy.  The  monkeys 
lost  all  sensation  to  pain  and  the  skin  for  a  radius  of  2-3  cm.  about  the  nodules 
was  distinctly  hypersensitive.  About  6-8  weeks  after  the  first  inoculation 
the  animals  exhibited  typical  signs  of  disseminated  infection  and  presented 
the  clinical  picture  of  human  leprosy  of  the  tuberculous  type.] 

Sugai  inoculated  an  emulsion  made  by  grinding  up  young  lepromata  in 
normal  saline  solution  into  Japanese  dancing  mice,  with  the  result  that  small 
granulomata  similar  to  those  of  miliary  tuberculosis  appeared  on  the  peri- 
toneum while  the  mesenteric  and  bronchial  glands  became  enlarged.  The 
leprosy  bacillus  was  present  in  the  lesions. 


350 


THE  LEPROSY  BACILLUS 


SECTION  II.—  MORPHOLOGY. 
1.  Microscopical  appearance. 

A.  In  human  lesions.  —  The  leprosy  bacillus  is  a  slender  rod-shaped  organism 
with   rounded    ends,    and    of   the    same  size  as  the 
^i  tubercle  bacillus  (5-6/x  x  0'5/x). 

Though  it  may  be  very  slightly  curved,  it  is  gener- 
*^     ally  speaking  straighter  than  the  tubercle   bacillus; 
occasionally  the  ends  are  slightly  swollen. 

Staining  reactions.  —  The  leprosy  bacillus,  like  the 
tubercle  bacillus,  stains  by  both  Ehrlich's  and  Ziehl- 
Neelsen's  methods,  but  is  more  acid-fast  and  therefore 
more  difficult  to  decolourize  than  the  tubercle  bacillus. 
^e  two  Bacilli  may  therefore  be  differentiated  by 
this  characteristic.  The  leprosy  bacillus  retains  the 

violet  in  Gram's  method. 

The   following  table   gives  the  differential  charac- 
teristics of  the  two  bacilli. 


-tf 


^  \ft 


FIG    213  —Bacillus    of 
leprosy  in  a  'film  fron^nasai 

met'hod.     xZio5o." 


LEPROSY  BACILLUS. 
Stains  with  aqueous  solutions  of  the 

basic  aniline  dyes. 
Stains  readily  by  Gram's  method. 

Stains  with  Ziehl-  Neelsen's  and  Ehr- 
lich's  solutions  and  resists  decolour- 
ization  for  a  long  time. 

Stains  by  Baumgarten's  method  (vide 

infra). 
Bacilli  present  in  very  large  numbers 

within  the  cells  of  the  leprous  nodule. 


TUBERCLE  BACILLUS. 
Does  not  stain  with  aqueous  dyes  con- 

taining  no  mordant. 
Stains  with  difficulty  by  Gram's  method 

(p.  307). 
Stains   with    Ziehl-Neelsen's   and    Ehr- 

lich's  solutions   but   is   much   more 

readily  decolourized  than  the  leprosy 

bacillus. 
Does     not     stain     by     Baumgarten's 

method. 
The  tubercle  cells  contain  only  a  few 

bacilli. 


Baumgarten's  method  of  staining.  —  Stain  for  5  minutes  in  the  cold  with 
aniline-violet,    decolourize    with    the 
following  solution  : 

Absolute  alcohol,     -         -     10  c.c. 

Nitric  acid,     -         -         -     1      ,. 

Wash  in  distilled  water.  Dry.  Mount. 

The  leprosy  bacillus  is  stained 
violet  :  the  tubercle  bacillus  is  de- 
colourized. 

Weil  has  shown  that  the  leprosy 
bacillus  only  stains  with  Ziehl-Neel- 
sen's and  Baumgarten's  methods 
when  taken  from  young  nodules. 
In  lesions  undergoing  resolution  these 
methods  as  well  as  Gram's  fail  to 
stain  the  bacillus. 

When  stained,  the  leprosy  bacillus 
is  often  granular  and  its  protoplasm 
contains  irregular  vacuoles.  The  ends 

are    often    swollen   and   Stain   easily    ,    FIG.  214  —Section  through  a  leprous  nodule  in  the 
,  ,  „.          v       larynx.     Carbol-fuchsm  and  methylene  blue.     (Oc. 

by  some  authors  these  swellings  are  2,  obj.  ^th,  Zeiss.) 
regarded  as  spores. 

Jamamoto's  stain.  —  By  staining  in  the  manner  now  to  be  described  Jama- 


MORPHOLOGY  351 

moto  claims  that  the  leprosy  bacillus  can  be  differentiated  from  the  tubercle 
bacillus  in  films. 

Fix  the  film  in  the  flame,  treat  for  10  minutes  in  a  bath  of  5  per  cent, 
solution  of  silver  nitrate  at  55°-60°  C.  and  then  transfer  to  the  reducing 
solution  : 

Pyrogallol,        -  2  -grams. 

Tannin,   -  1  gram. 

Distilled  water,  -         100  c.c. 

Tubercle  bacilli  are  stained  black  :  leprosy  bacilli  are  unstained  and  may 
be  counter-stained  by  carbol-fuchsin. 

[B.  In  cultures. — Rost  found  acid-fast  bacteria  massed  together  in  parallel 
arrangement  in  his  cultures  from  leprous  nodules  on  milk-fish  broth.  When 
sub-cultivated  on  agar  and  broth  a  feebly  acid-fast  bacillus  developed  and  it 
was  found  that  the  acid-fastness  could  be  increased  by  growing  the  organism 
on  milk.  The  organism  is  highly  pleomorphic.  In  sub-cultures  after  48 
hours'  incubation  the  appearance  is  the  same  as  in  the  nodules  of  a  leper. 
In  older  cultures  or  when  grown  under  unfavourable  conditions  "  degenerate 
forms  are  found  which  double  or  treble  their  usual  length  with  a  moniliform 
arrangement  and  lose  their  acid-fastness.  These  break  down  after  a  few 
days  into  clumps  of  small  acid-fast  coccoid  forms." 

[Bayon  cultivated  from  cases  of  human  leprosy  an  acid-resisting  diphtheroid 
organism  which  acquired  acid-fast  properties  on  being  inoculated  into  rats 
and  mice. 

[Williams  grew  a  very  pleomorphic  streptothrix  from  the  lesions  of  human 
leprosy  "  which  in  addition  to  changes  in  form  exhibited  marked  changes 
in  its  staining  reactions  in  regard  to  the  quality  known  as  acid-fastness." 
Williams  describes  the  following  forms  : 

(a)  On  broth  media  and  on  potato-broth  a  non-acid-fast  streptothrix  in  the 
mycelial  stage  which  produced  acid-fast  rods. 

(ft)  On  milk  and  lemco-broth  a  non-acid-fast  diphtheroid  bacillus  which  also 
produced  acid -fast  rods. 

(y)  On  Rost's  medium  an  acid-fast  bacillus  which  is  but  the  broken-down  stage 
of  a  streptothrix,  and 

(8)  On  Dorset's  egg  medium  an  acid-fast  mycelium.  This  streptothrix  which 
was  cultivated  from  a  leper  passed  through  respectively  all  the  stages  described 
above.  ] 

2.  Cultures. 

Very  little  is  yet  known  about  the  cultivation  of  the  leprosy  bacillus. 
Roux,  Cornil,  and  Chantemesse  failed  in  their  attempts  to  grow  the  bacillus  : 
numerous  observers  have  obtained  cultures  after  sowing  pieces  of  leprous 
tissues,  but  in  the  great  majority  of  cases  these  were  cultures  of  organisms 
of  secondary  infection  and  not  cultures  of  the  leprosy  bacillus  (vide  infra). 

Bordoni-Uffreduzzi  for  example  described  certain  growths  as  cultures  of  the 
leprosy  bacillus  which  were  obviously  cultures  of  the  tubercle  bacillus  :  similarly 
Neisser's  cultures  were  not  cultures  of  the  leprosy  bacillus.  Babes'  cultures  were 
cultures  of  a  bacillus  which  did  not  stain  either  with  Ehrlich's  or  Ziehl-Neelsen's 
stain  :  apparently  also  Ducrey's  anaerobic  organism  may  be  dismissed  without 
consideration. 

Czaplewski,  Spronck,  Teich,  Levy,  Rost  and  others  seem  to  have  grown 
cultures  of  the  leprosy  bacillus,  though  it  is  to  be  noted  that  the  descriptions 
of  their  cultures  do  not  at  all  coincide. 

Spronck  sowed  leprous  bone  marrow  and  non-ulcerated  leprous  nodules  on  neutral 
glycerin-potato  :  the  growth  was  said  to  have  been  sub -cultivated  on  coagulated 
serum,  glucose-glycerin-agar  and  glycerin- fish -broth  (p.  319)  but  not  on  glycerin- 
potato.  Growth  took  place  at  25°  C.  and  was  copious  at  37°  C. 


352  THE   LEPROSY  BACILLUS 

On  glycerin-potato  (primary  culture). — After  incubation  for  10  days  at  37°  C. 
very  small,  yellowish,  hardly  visible  colonies  appeared. 

On  glucose-glycerin-agar. — Small,  colourless,  irregularly  circular  colonies. 

On  coagulated  serum. — Small,  greyish-yellow,  irregularly  circular  colonies. 

On  fish  broth. — A  viscous  precipitate  adhering  to  the  sides  of  the  vessel. 

Bacilli  from  these  cultures  were  agglutinated  by  the  blood  of  lepers  in  dilutions 
of  1  in  70  to  1  in  1000  and  only  in  dilutions  of  1-30  or  1-40  by  the  blood  of  other 
persons. 

[Rost  employed  a  medium  of  the  following  composition  : 

Distilled  volatile  alkaloid  of  rotten  fish,       -  250  c.c. 

Weak  Lemco  broth  (without  peptone  or  salt),      -         -         -         250  c.c. 
Milk,  50  c.c. 

which  he  sowed  with  material  from  cases  of  nodular  leprosy  and  obtained  in  3  days 
a  slight  stringy  growth  at  the  bottom  of  the  tube  which  on  microscopical  examination 
proved  to  be  masses  of  acid-fast  bacteria.  Sub-cultures  were  sown  on  agar  and 
in  broth  (no  salt  or  peptone)  and  a  growth  was  obtained  in  48  hours. 

[Clegg  grew  the  parasite  of  leprosy  symbiotically  with  amoebae  and  their  symbiotic 
bacteria  on  an  agar  medium.  After  destroying  the  amcebse  and  bacteria  by  heat 
at  60°  C.  for  half  an  hour  pure  cultures  of  the  organism  were  obtained  by  sub -cul- 
tivating on  ordinary  media — agar,  potato,  milk,  etc. 

[Duval  used  egg-albumin  or  human  blood  serum  poured  into  sterile  Petri 
dishes  and  inspissated  for  3  hours  at  70°  C. 

[The  excised  leprous  nodule  is  cut  into  thin  slices  ('5-1  mm.)  and  distributed  over 
the  surface  of  the  albumin.  After  sowing,  the  medium  is  bathed  in  a  1  per  cent. 
solution  of  trypsin  added  with  a  pipette  but  the  tissue  must  not  be  submerged. 
Incubate  at  20°  C.1  for  a  week  to  10  days  trypsin  being  added  from  time  to  time 
as  evaporation  necessitates. 

[Sub-cultures  may  be  sown  on  the  albumin- trypsin  medium  but  after  sub- 
cultivating  three  or  four  times  growth  can  be  obtained  on  a  glycerin- agar. 

Agar. 20  grams. 

Salt,         -         -  3      „ 

Glycerin,  30  c.c. 

Distilled  water,  -         500  c.c. 

Mix,  clear  and  sterilize  in  the  usual  manner. 

To  10  c.c.  of  the  agar  at  42°  C.  add  5  c.c.  of  unheated  turtle  muscle  infusion  : 
Turtle  muscle  cut  into  fine  pieces,       -  500  grams. 

Water,     -         ...  -         500  c.c. 

Keep  in  the  ice  chest  for  48  hours :  filter  through  gauze  and  then  through  a  Berkefeld 
filter. 

[Duval  claims  that  the  organism  he  cultivated  was  the  true  leprosy  parasite 
because  with  its  aid  he  was  able  to  produce  the  lesions  of  leprosy  in  a  monkey. 

[Twort  has  introduced  a  method  of  cultivation  based  upon  the  addition  of 
sterilized  tubercle  bacilli  to  an  egg  medium.  Growth  is  not  visible  to  the 
naked  eye  for  about  6  weeks. 

[The  material  used  was  the  nasal  discharge  and  scrapings  from  a  typical  leper. 

[The  nasal  discharge  was  first  placed  in  a  2  per  cent,  solution  of  ericolin — a  gluco- 
side — at  38°  C.  for  1  hour  to  destroy  contaminating  organisms  and  the  sediment 
was  then  used  for  sowing  the  culture  medium. 

[The  culture  medium. — Cultivations  of  the  tubercle  bacillus  on  Dorset's  egg  medium 
were  steamed  and  the  growth  scraped  off  (care  being  taken  to  avoid  the  medium 
containing  the  waste  products).  The  tubercle  bacilli  were  ground  up  into  an  emul- 
sion with  glycerin  and  normal  saline  solution,  steamed  for  30  minutes  and  added 
to  the  yolk  and  white  of  new-laid  eggs  in  the  following  proportions : 

Eggs. 75  parts. 

8  per  cent,  saline  solution,  95  parts. 

Mix  well  and  add  tubercle  bacilli  1  per  cent,  and  glycerin  5  per  cent,  or  less. 

[The  medium  was  tubed,  heated  to  60°  C.  for  1  hour  and  on  the  following  morning 

[l  In  his  original  experiments  Duval  incubated  at  37°  C.  but  now  finds  room  tempera- 
ture more  suitable.] 


SERUM  THERAPY  353 

incubated  at  38°  C.  for  6  hours  after  which  it  was  again  heated  in  a  water  bath  at 
60°  C.  for  1  hour  and  then  sloped  at  85°  C. 

[The  ericolinized  nasal  discharge  was  sown  and  the  tubes  capped  and  incubated 
at  38°  C.  After  24  hours  the  medium  had  absorbed  a  quantity  of  the  ericolin  so 
the  material  was  transferred  to  other  tubes. 

[The  bacilli  grew  and  were  sub-cultivated  in  pure  culture.  In  sub-cultures  the 
bacilli  were  long,  thin,  beaded  rods,  well  formed  and  quite  acid-fast.  Growth  appears 
as  a  thin  colourless  film  visible  to  the  naked  eye  in  6  weeks.  ] 

[Bayon  states  that  the  most  favourable  media  appear  to  be  either  placental- 
extract-glycerin-agar,  or  horse-serum-nutrose-agar  containing  2  per  cent, 
ground-up  smegma  bacilli  (Twort's  method).  From  the  nodules  of  a  case  of 
leprosy  this  observer  isolated  an  organism  which  "  grew  rapidly  as  a  white 
viscid  growth  on  placental-extract-glycerin-agar."  Morphologically  it  assumes 
one  of  three  forms  :  (i)  a  non-acid-fast  and  non-acid-resisting  streptothrix, 
(ii)  a  pleomorphic.  acid-resisting  diphtheroid  bacillus  and  (iii)  a  definitely 
acid-fast  bacillus  indistinguishable  from  the  bacillus  in  the  tissues. 

[The  organism  appears  to  be  identical  with  that  cultivated  from  leprous 
lesions  by  Kedrowsky  so  long  ago  as  1901  and  this  latter  Bayon  regards  as 
the  true  parasite  of  leprosy  for  the  following  reasons  :  it  has  been  cultivated 
repeatedly  from  lepers,  it  causes  leprous  lesions  in  rats  and  mice,  it  reacts 
specifically  with  the  serum  of  lepers  in  a  way  that  neither  human  nor  avian 
tubercle  bacilli  react  and  lastly  it  is  not  identical  with  any  other  known 
organism.  ] 

According  to  Ch.  Nicolle  and  Weil,  primary  cultures  of  the  leprosy  bacillus 
can  be  obtained  by  sowing  non-ulcerated  leprous  tissue  rich  in  bacilli  in 
considerable  amount  in  the  water  of  condensation  of  glucose-glycerin-agar 
tubes  to  which  serum  may  or  may  not  have  been  added  ;  the  bacilli  appear 
to  grow  solely  at  the  expense  of  the  cells  of  the  material  sown  and  cannot  be 
sub-cultivated.  Weil  has  also  succeeded  in  growing  cultures  by  sowing  the 
material  in  the  yolk  of  the  whole  egg  and  on  yolk  of  egg-agar  (p.  53,  A)  ;  but 
here  again  sub-cultures  could  not  be  obtained. 

SECTION  III.— SERUM  THERAPY. 

Carasquilla  was  the  first  to  attempt  the  preparation  of  an  antiserum  by 
inoculating  large  animals  with  the  blood  and  serum  of  leper  patients.  Later, 
Laverde  injected  asses,  lambs  and  horses  with  blood  and  serum  from  leprosy 
patients,  with  the  juice  of  lepromata  and  even  with  the  pulp  of  an  epithelioma 
of  the  cervix  uteri :  the  serum  of  the  treated  animals  had  a  favourable 
influence  on  the  course  of  the  disease  (10-20  c.c.  were  used  for  inoculation). 
Laverde's  results  were  confirmed  by  Buzzi,  Abraham,  and  Arning ;  Hallo- 
peau,  Neisser,  and  Brieger,  however,  failed  to  obtain  similarly  favourable 
results  with  the  antiserum. 

Metchnikoff  and  his  pupils  showed  that  the  serum  prepared  by  Laverde 
contained  neither  leprous  products  nor  toxin,  but  that  the  inoculation  of 
serum,  blood,  or  cellular  elements  of  one  animal  into  an  animal  of  another 
species  leads  to  the  formation  in  the  latter  of  substances  (cytotoxins}  which 
have  the  property  of  destroying  the  cells  of  the  animal  from  which  the  material 
for  inoculation  was  taken  :  and  they  demonstrated  that  the  improvement 
noted  in  the  lepers  treated  with  Laverde's  serum  should  be  attributed  to 
these  cytotoxins.  The  inconstant  results  are  explicable  on  the  ground  of  the 
delicate  nature  of  the  cytotoxins,  since  these  are  destroyed  by  transport,  the 
addition  of  carbolic  acid,  etc. 

Metchnikoff  and  Besredka  inoculated  a  goat  over  a  period  of  36  days  with  34  c.c. 
of  defibrinated  blood  from  a  healthy  man.  The  goat's  serum  acquired  powerful 

Z 


354  THE   LEPROSY   BACILLUS 

agglutinating  and  haemolytic  properties  for  human  blood :  a  given  volume  of  the 
serum  agglutinated  at  once  and  dissolved  in  7  minutes  all  the  red  cells  in  an  equal 
volume  of  human  blood. 

When  injected  into  lepers  in  doses  of  1,  3,  and  7  c.c.  this  serum  to  some  extent 
relieved  pain,  and  caused  congestion  and  suppuration  of  some  of  the  lepromata 
with  the  result  that  sloughs  formed  which  afterwards  became  detached :  in  a  few 
cases  an  insignificant  febrile  attack  was  noticed.  In  short,  the  results  of  using 
Metchnikoff's  serum,  although  not  so  good,  were  very  similar  to  those  obtained 
with  the  serum  prepared  by  Carasquilla,  Laverde,  and  others. 

In  Metchnikoff's  opinion  the  favourable  results  following  the  use  of  such  serums 
should  be  attributed  to  the  leucotoxin  developed  in  response  to  the  inoculation  into 
the  tissues  of  an  animal  of  human  leucocytic  products  ;  this  leucotoxin  should  in 
suitable  doses  lead  to  stimulation  of  the  leucocytic  system  :  the  haemotoxin  is  of 
no  therapeutic  value,  and  indeed  prevents  the  employment  of  sufficiently  large 
doses  of  serum.  The  obvious  conclusion  from  this  argument  is  that  in  the  treat- 
ment of  leprosy  an  attempt  should  be  made  to  prepare  an  antiserum  by  inoculating 
an  animal  with  blood  serum  alone  or  better  still  with  human  lymphatic  glands. 

SECTION  IV.— DETECTION  AND   IDENTIFICATION   OF   THE 
LEPROSY   BACILLUS. 

Microscopical  examination  is  at  present  the  only  means  of  detecting  the 
leprosy  bacillus.  Sections  should  be  cut  and  films  made  of  the  suspected 
tissues  and  fluids.  [Cultures  should  however  be  attempted.] 

The  bacillus  of  leprosy  is  found  in  the  leprous  nodules,  bone  marrow, 
and  spleen.  The  bacillus  can  also  be  found  in  the  glands,  in  the  swellings 
along  the  nerves,  in  the  discharge  from  ulcerating  lesions,  in  the  saliva  when 
the  buccal  mucous  membrane  is  affected,  in  the  stools  when  the  disease  infects 
the  large  intestine,  in  the  secretion  of  the  testicle  when  that  organ  is  involved, 
in  the  milk  (Babes),  etc.  Sticker  has  drawn  attention  to  the  presence  of  the 
bacillus  in  the  nasal  mucus.  Nasal  lesions  are  commonly  present  from  the 
early  stages  of  the  disease  and  were  found  in  128  out  of  153  lepers  examined 
by  Sticker  ;  examination  of  films  of  the  nasal  mucus  will  therefore  often 
afford  confirmation  of  the  diagnosis.1 

Leprous  nodules  consist  of  large  cells,  similar  to  epithelioid  cells,  having 
as  a  rule  a  single  nucleus  and  crammed  full  of  bacilli :  these  constitute  the 
lepra  cells.  The  leprosy  bacillus  is  therefore  intra-cellular. 

During  life  portions  of  a  leproma  can  be  easily  excised,  since  it  is  known  that 
in  the  majority  of  these  lesions  there  is  an  absence  of  all  sensation.  Manson  recom- 
mends isolating  a  succulent  leproma  in  a  pile  clamp,  slowly  screwing  up  the  jaws 
of  the  instrument  so  as  to  drive  out  the  blood,  pricking  the  now  pallid  leproma  and 
then  collecting  on  a  cover-glass  the  droplet  of  "  leper  juice  "  which  exudes  from 
the  puncture. 

Arning  has  never  found  the  bacillus  in  the  blood.  According  to  Cornil, 
Babes  and  Goujerot  the  bacillus  enters  the  blood  stream  a  few  days  before 
death  and  especially  during  the  febrile  attacks. 

The  following  technique  is  recommended  for  the  detection  of  the  bacillus  : 
(a)  Stain  films  by  Ziehl-Neelsen's  method.      The  leprosy  bacillus  is  dif- 
ferentiated from  the  tubercle  bacillus  by  three  tests — 

1.  Simple  staining  with  a  watery  alcoholic  solution  of  fuchsin  ; 

2.  Gram's  stain ; 

3.  Baumgarten's  method  of  staining. 

1  In  examining  films  of  the  nasal  mucus  care  must  be  taken  to  distinguish  the  leprosy 
bacillus  from  the  bacillus  of  Karlinski.  The  latter  bacillus  is  found  in  the  nasal  mucus  of 
man  quite  apart  from  leprosy  or  tuberculosis  :  it  gives  rise  to  no  symptoms,  morphologi- 
cally resembles  the  bacillus  of  leprosy,  and  is  acid-fast.  In  cultures,  it  grows  easily 
on  ordinary  media,  and  is  pathogenic  to  guinea-pigs  when  inoculated  intra-peritoneally. 


ASSOCIATED  MICRO-ORGANISMS  355 

(b)  Stain  sections  of  tissues  hardened  in  alcohol  and  embedded  in  paraffin 
by  Ziehl-Neelsen's  method  :  if  necessary,  the  differential  tests  given  above 
can  be  applied. 

A  good  diagnostic  point  is  afforded,  as  has  been  said,  by  the  enormous 
numbers  of  bacilli  to  be  seen  in  the  lepra  cells  :  tubercle  cells  never  contain 
more  than  a  few  bacilli.  Finally,  the  inoculation  of  a  guinea-pig  [will  exclude 
tuberculosis]. 

Sub-cutaneous  inoculation  of  tuberculin  produces  a  reaction  in  persons 
suffering  from  leprosy  while  the  ophthalmo-reaction  is  negative  (Nicolle  and 
Uriarti,  Gaucher  and  Abrami). 

Associated  micro-organisms. 

The  lesions  of  leprosy  being  so  frequently  situated  in  the  skin  and  mucous 
membranes  or  in  the  lungs  are  particularly  liable  to  secondary  infection. 

Lesions  of  the  skin  and  mucous  membranes  are  very  soon  invaded  by  the  ordinary 
organisms  of  suppuration  (staphylococci,  bacillus  pyocyaneus,  etc.).  In  the  case  of 
a  leper  in  Tunis  the  author  was  able  to  demonstrate  in  the  discharge  from  the  specific 
lesions  in  addition  to  a  few  leprosy  bacilli,  staphylococcus  aureus,  bacillus  pyocyaneus 
and  bacillus  coli.  These  organisms  of  secondary  infection  may  invade  the  tissues 
in  cases  of  leprosy  and  give  rise  to  a  rapidly  fatal  pyaemia  (Babes). 

Babfes  has  frequently  found  the  tubercle  bacillus  in  persons  suffering  from  leprosy, 
and  the  two  organisms  are  frequently  found  in  association  especially  in  the  lung : 
and  in  pulmonary  lesions  the  pneumococcus  may  also  be  present. 

In  three  cases  of  leprosy,  Babes  found  as  a  secondary  infection  in  the  bone  marrow, 
spleen,  and  kidneys,  a  bacillus  which  was  easily  cultivated  outside  the  body  and  did 
not  stain  by  Ehrlich's  or  Ziehl's  methods. 


CHAPTER  XX. 
BACILLUS  DYSENTERIC  EPIDEMICS. 

Introduction. 

Section  L — Experimental  inoculation,  p.  357. 

1.  Shiga  bacillus,  p.  357.     2.  Flexner  bacilli,  p.  358. 
Section  II. — Morphology,  p.  358. 

1.  Microscopical  appearance  and  staining  reactions,  p.  358.     2.  Cultural  charac- 
teristics, p.  359. 
Section  III. — Biological  properties,  p.  359. 

1.  Biochemical  reactions,  p.  359.  2.  Vitality,  p.  360.  3.  Toxin;  p.  361.  4.  Vaccina- 
tion and  serum  therapy,  p.  361.  5.  Agglutination,  p.  363.  6.  Precipitins,  p.  363. 
7.  Immune  body,  p  363. 

Section  IV. — Detection,  isolation  and  identification  of  the  dysentery  bacilli,  p.  364. 
Serum  diagnosis,  p   364. 

The  bacillus  dysentericus  El  Tor  No.  1,  p.  365. 

THE  bacillus  of  epidemic  dysentery  was  discovered  by  Chantemesse  and 
Widal  in  1888  :  Shiga  amplified  their  observations  and  adduced  additional 
evidence  in  proof  of  the  specific  relationship  of  the  bacillus  to  the  disease. 

The  term  dysentery  is  applied  to  a  clinical  syndrome  indicating  certain  lesions  of 
the  large  intestine  and  aetiologically  includes  two  distinct  diseases,  one  caused  by 
an  amoeba  and  the  other  by  a  bacillus.  The  former  is  an  endemic  disease  of  warm 
climates  and  is  frequently  complicated  by  abscess  of  the  liver :  the  latter  is  an 
epidemic  disease  not  complicated  by  abscess  of  the  liver  and  prevalent  both  in  warm 
and — especially — in  temperate  climates. 

It  is  possible  that  in  rare  cases  symptoms  of  dysentery  may  be  due  to  certain  other 
parasites  which  up  till  now  have  received  little  attention,  such  for  example  as  Balan- 
tidium  coli,  Spirilla,  or  Trichomonas. 

[Dysentery  bacilli  have  been  isolated  from  a  number  of  cases  of  infantile  diarrhoaa 
(quite  unrelated  to  any  epidemic  of  dysentery)  by  Bassett  and  Duval  in  the  United 
States ;  and  in  South  Africa,  Birt  found  dysentery  bacilli  in  7  out  of  10  cases  of 
this  disease.  In  London,  however,  Morgan  failed  to  find  any  bacilli  of  the  dysentery 
group  in  cases  of  infantile  diarrhoea. 

[Asylum  dysentery  has  been  shown  to  be  a  bacillary  dysentery  and  bacilli  of  the 
dysentery  group  have  been  isolated  from  cases  of  the  disease  in  England,  Germany 
and  America  (Eyre  ;  Aveline,  Boycott  and  W.  F.  Macdonald  ;  Kruse  ;  Vedder  and 
Duval). 

[Sporadic  cases  of  dysentery  are  said  to  occur  in  England  though  rarely  (Marshall ; 
Bainbridge  and  Dudfield).  Ledingham's  investigations  would  appear  to  show  that 
dysentery  bacilli  are  occasionally  found  in  the  stools  of  healthy  persons.  ] 

In  patients  suffering  from  bacillary  dysentery  the  organism  is  found  in 
large  numbers  in  the  intestinal  mucous  membrane  and  in  the  stools  especi- 
ally in  the  mucous  flakes.  It  does  not  become  generalized,  and  with  the 


EXPERIMENTAL  INOCULATION  357 

exception  of  one  case  recorded  by  Rosenthal  the  organism  has  never  been 
found  in  the  blood  stream  :  according  to  some  observers  it  is  occasionally 
present  in  the  mesenteric  glands  (Shiga,  Duval  and  Bassett,  [Aveline,  Boycott 
and  Macdonald]). 

[Aveline,  Boycott  and  Macdonald  isolated  the  organism  from  the  spleen 
in  one  out  of  three  fatal  cases  of  asylum  dysentery.  ] 

Several  varieties  of  dysentery  bacilli  have  been  described,  differing  from 
one  another  in  one  or  more  particulars  and  especially  in  their  action  upon 
sugars.  For  practical  purposes  these  varieties  may  be  divided  into  two 
types  :  the  Shiga-Kriise  or  non-mannite  fermenting  type  and  the  Flexner 
or  mannite  fermenting  type  (see  also  p.  360). 

Some  authors  regard  the  differences  between  these  two  types  as  sufficient 
to  justify  their  classification  as  separate  species.  But  it  is  held  that  these 
differences  are  not  marked  enough  to  warrant  so  complete  a  separation,  and 
the  view  put  forward  by  Gay  and  Duval  which  is  perhaps  of  the  nature  of  a 
compromise,  commands  general  acceptance.  These  authors  consider  that 
the  bacilli  causing  bacillary  dysentery  are  to  be  regarded  as  belonging  to  a 
group  of  organisms  exhibiting  certain  variations  among  themselves  rather 
than  as  a  single  sharply-defined  species.  There  is  a  similar  multiplicity  of 
varieties  of  the  causal  organism  of  cholera,  as  will  be  shown  later. 

The  Shiga  type  of  bacillus  is  the  common  cause  of  dysentery,  and  it  will 
be  therefore  described  at  length  in  the  following  pages,  the  points  of  difference 
between  it  and  the  Flexner  type  being  noted  in  the  proper  places.  The 
general  statement  may  here  be  made  that  all  strains  of  Shiga 's  bacillus  agree 
in  their  cultural  and  other  characteristics,  while  under  the  title  of  Flexner's 
bacillus  a  number  of  very  closely  related  though  not  absolutely  identical 
organisms  are  included. 

SECTION   I.— EXPERIMENTAL   INOCULATION. 

Shiga  bacillus. 

Speaking  generally,  the  Shiga  type  of  bacillus  is  much  more  highly  patho- 
genic to  laboratory  animals  than  are  bacilli  of  the  Flexner  type. 

Infection  by  the  alimentary  canal.  In  man. — Strong  and  Musgrave  after 
administering  some  bi-carbonate  of  soda  to  a  condemned  criminal  gave  him 
a  two-day-old  broth  culture  of  the  dysentery  bacillus.  After  an  incubation 
period  of  36  hours  the  man  suffered  from  a  typical  attack  of  dysentery  with 
blood-stained  stools  from  which  he  made  a  rapid  recovery.  The  bacillus  was 
isolated  from  the  stools. 

In  animals. — Infection  of  the  alimentary  canal  whether  by  feeding  or 
inoculation  generally  gives  negative  results  (Rosenthal,  Shiga,  Conradi,  and 
others). 

After  feeding  guinea-pigs  with  dysentery  bacilli,  however,  Chantemesse  found 
lesions  in  the  intestines  similar  to  those  seen  in  human  dysentery ;  and  Shiga,  after 
introducing  a  culture  into  the  stomach  of  a  cat,  noticed  that  it  suffered  from  mucous 
diarrhoea  and  found  the  bacilli  in  increased  numbers  in  the  stools.  Kazarinow 
introduced  very  large  quantities  of  culture  into  the  intestines  of  rabbits  by  means 
of  an  cesophageal  sound,  and  post  mortem  found  characteristic  lesions  in  the 
intestine. 

Intra-peritoneal  inoculation. — Inoculation  of  dysentery  bacilli  into  the 
peritoneal  cavity  is  rapidly  fatal  to  most  animals.  Post  mortem,  the  peri- 
toneal cavity  contains  a  blood-stained  serous  exudate  and  the  intestine  is 
very  markedly  hypersemic  but  presents  none  of  the  lesions  characteristically 
seen  in  the  human  disease. 


358  THE   DYSENTERY   BACILLUS 

Intra- venous  inoculation. — In  the  majority  of  animals  death  from  septi- 
caemia rapidly  follows  the  inoculation  of  bacilli  into  the  veins.  Post  mortem, 
the  intestine  is  found  to  be  slightly  hyperaemic. 

Sub-cutaneous  inoculation. — The  most  interesting  experimental  results  are 
obtained  by  inoculating  animals  sub-cutaneously  :  in  rabbits,  dogs,  cats 
and  young  pigs  such  inoculation  is  followed  by  lesions  similar  to  those  found 
in  the  human  subject.  Guinea-pigs  are  less  susceptible  than  other  laboratory 
animals  to  this  method  of  infection. 

Rabbits. — Sub-cutaneous  inoculation  of  3-4  c.c.  of  a  broth  culture  is  fatal 
in  4-6  days. 

Inoculation  leads  first  of  all  to  the  formation  of  a  large  inflammatory  oedema 
at  the  site  of  inoculation  and  this  is  soon  followed  by  a  rise  of  temperature  and  the 
onset  of  diarrhoea,  then  paralysis  of  the  hind  limbs  appears  and  finally  the  tem- 
perature begins  to  fall  and  continues  to  decline  steadily  until  death  occurs.  Post 
mortem,  lesions  are  present  throughout  the  alimentary  canal  being  especially  marked 
in  the  colon.  In  that  portion  of  the  intestine  the  mucous  membrane  is  thickened, 
swollen,  intensely  hypersemic  and  covered  with  blood-stained  mucus ;  small  foci 
of  superficial  necrosis  and  haemorrhagic  patches  are  also  seen,  the  former  occasionally 
ending  in  ulceration.  The  bacillus  multiplies  both  in  the  mucus  and  in  the  mucous 
membrane  where  it  is  found  in  pure  culture. 

Dogs. — A  "  true  representation  of  human  dysentery  with  its  painful  and 
frequent  strainings,  characteristic  stools  and  lesions  "  is  seen  in  young  dogs 
as  a  result  of  sub-cutaneous  inoculation  (Vaillard  and  Dopter). 

Following  the  inoculation  of  one  or  two  agar  cultures,  the  temperature  rises  and 
an  cedematous  infiltration  appears  at  the  site  of  inoculation :  the  animal  lies  down, 
seems  ill  and  is  apparently  in  pain,  and  the  motions  become  frequent  and  in  character 
similar  to  those  of  human  dysentery.  These  symptoms  are  followed  by  wasting 
and  a  fall  of  temperature  to  below  normal,  and  death  takes  place  between  the  third 
and  sixth  day.  Post  mortem,  lesions  similar  to  those  described  in  the  case  of  the 
rabbit  are  found  in  the  intestine  which  is  also  extensively  ulcerated,  and  the  mes- 
enteric  glands  are  swollen.  The  bacillus  is  present  in  pure  culture  in  the  affected 
parts  of  the  mucous  membrane. 

Young  pigs. — Sub-cutaneous  inoculation  in  these  animals  generally  leads 
to  a  fatal  attack  of  dysentery.  Post  mortem,  lesions  are  found  resembling 
those  in  the  human  disease. 

Note. — After  sub-cutaneous  inoculation  the  bacillus  can  always  be  found  in  the 
local  lesion  at  the  site  of  inoculation  and  frequently  also  in  the  mesenteric  glands, 
but  only  exceptionally  in  the  spleen  and  liver  and  never  in  the  blood  of  the  heart. 

Bacilli  of  the  Flexner  type. 

Bacilli  of  the  Flexner  type  are  far  less  pathogenic  than  the  Shiga  bacillus. 
Intra-venous  inoculation  is  not  followed  by  severe  symptoms  and  rarely 
leads  to  death  in  the  case  of  dogs  and  rabbits  :  intra-peritoneal  inoculation 
is  more  dangerous  and  produces  a  fatal  peritonitis  in  guinea-pigs  :  feeding 
experiments  give- negative  results.  Sub-cutaneous  inoculation  is  not  followed 
by  the  symptoms  and  lesions  of  experimental  dysentery  and  does  not  lead  to 
a  fatal  result ;  after  a  marked  local  reaction  the  animal  recovers. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  dysentery  bacillus  is  a  small  rod-shaped  organism  morphologically 
like  the  colon  bacillus  ;  it  measures  1-3/u  long.  In  cultures  the  organism  is 
pleomorphic,  and  long  almost  filamentous  forms  are  found  side  by  side  with 
very  short  bacilli.  It  does  not  form  spores,  and  is  non-flagellated  and  non- 


MORPHOLOGY  359 

motile,  though  it  exhibits  oscillatory  movements  which  have  been  compared 
to  those  of  a  compass  needle. 

Staining  reactions. — The  dysentery  bacillus  stains  with  the  ordinary  basic 
aniline  dyes,  and  with  weak  dyes  tends  to  stain  more  deeply  at  the  ends  than 
in  the  centre.  It  is  gram-negative. 

Films  should  be  stained  with  carbol-thionin  or  carbol-methylene-blue  : 
sections  with  thionin  or  by  Nicolle's  tannin  method  (p.  217). 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  dysentery  bacillus  grows  on  all  the  ordinary 
alkaline  media,  and  equally  well  under  aerobic  or  anaerobic  conditions.  The 
optimum  temperature  is  37°  C.,  though  growth  takes  place  within  wide 
limits  (10°-40°  C.). 

Characteristics  of  growth.  Broth. — Growth  is  visible  after  incubating  for 
6  hours,  and  after  24  hours  the  medium  is  uniformly  cloudy  and  has  a  watered- 
silk  appearance.  On  further  incubation  a  small  glutinous  deposit  forms 
which  continues  to  increase  until  towards  the  end  of  the  second  day,  when  the 
upper  part  of  the  broth  is  clear  :  no  pellicle  is  formed  on  the  surface.  The 
cultures  have  a  peculiar  spermatic  odour. 

Gelatin. — The  'growth  is  like  that  of  the  typhoid , 
bacillus.  Isolated  colonies  are  small,  delicate  andf 
translucent  with  edges  like  the  edges  of  a  vin< 
leaf.  In  stroke  culture  the  growth  consists  of  al 
thin  narrow  opalescent  band.  The  medium  is  not 
liquefied. 

Agar. — On  agar  the  growth  resembles  that  of 

+Tio  •HrrVhr.irl  Knr>illn«  FlG-  215.— Bacillus  dysenterice. 

tne  typnoid  DaClllUS.  Potato  culture,  6  days. 

Potato.— The   growth  on  potato  consists  of  a 

moist,  shiny  glaze  which  is  at  first  so  scanty  as  to  be  hardly  visible.  Later 
it  acquires  a  greyish  or  yellowish  tint. 

Milk. — Milk  is  not  coagulated. 

Bacilli  of  the  Flexner  type. 

Cultures  of  bacilli  of  the  Flexner  type  have  the  same  characteristics  as 
but  are  more  luxuriant  than  those  of  the  Shiga  bacillus.  In  broth,  after 
incubating  for  about  3  days  a  ring  of  growth  adherent  to  the  sides  of  the  tube 
is  formed  on  the  surface  of  the  medium  ;  this  falls  to  the  bottom  a  few  days 
later. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Biochemical  reactions. 

Action  on  carbohydrates. — The  Shiga  bacillus  does  not  ferment  carbo- 
hydrates. The  blue  colour  of  litmus  milk  is  unchanged,  but  litmus  whey  is 
slightly  reddened  at  first  becoming  blue  again  about  the  second  or  third  day. 
No  gas  is  formed  in  litmus-lactose-broth.  Neutral-red  in  glucose  media  is 
not  reduced. 

Bacilli  of  the  Flexner  type. — These  bacilli  ferment  mannite  and  maltose 
but  no  other  sugar  [but  see  below]  :  litmus  media  containing  mannite  and 
maltose  are  turned  red,  but  no  gas  is  formed.  In  lactose  media  they  behave 
like  the  Shiga  bacillus. 

[All  strains  of  the  Flexner  or  mannite-fermenting  type  apparently  produce 
acid  in  mannite,  glucose,  galactose,  arabinose,  and  raffinose  :  many  ferment 
(acid,  no  gas)  maltose  and  dextrin  though  not  necessarily  both,  while  a  few 


360  THE   DYSENTERY   BACILLUS 

form  acid  in  sorbite,  imilin,  salicin,  and  isodulcite.  One  strain  of  the  Flexner 
type — apparently  identical  with  the  dysentery  bacillus  isolated  by  Strong — 
forms  acid  in  dulcite  and  cane  sugar  (Morgan). 

[Hiss  divided  dysentery  bacilli  into  four  groups  according  to  their  fermentation 
reactions. 

[The  first  or  Shiga  group  ferments  the  monosaccharides  and  sometimes,  after  an 
interval  of  several  days,  maltose. 

[The  second  group  represented  by  Hiss'  Y  bacillus  (isolated  from  dysenteric 
diarrhoea  in  children  due  to  milk  and  identical  with  the  bacillus  found  in  cases  of 
asylum  dysentery  by  Kruse  and  others)  ferments  the  monosaccharides  and  mannite. 
Maltose  and  saccharose  are  sometimes  also  but  with  difficulty  decomposed  with  the 
formation  of  acid. 

[The  third  group  consisting  of  Strong's  Philippine  bacillus  ferments  the  mono- 
saccharides, mannite  and  saccharose,  occasionally  also  maltose. 

[The  fourth  or  Flexner  (Manilla)  group  decomposes  the  monosaccharides,  mannite 
maltose,  saccharose  and  dextrin. 

[To  these  Shiga  subsequently  added  a  fifth  group  ;  the  characteristic  of  the 
organisms  comprising  it  being  that  they  give  first  of  all  an  acid  reaction  in  mannite, 
which  subsequently  changes  to  an  alkaline  reaction. 

[Aveline,  Boycott  and  Macdonald  find  that  the  fermentative  reactions  of  the 
Flexner  group  towards  maltose  and  cane  sugar  are  variable.  Thus,  24  cultures  were 
tested  with  the  result  shown — 

CANE  SUGAR.  MALTOSE. 

Days  incubated,      -         -         -         1     7     14    28  1     7     14     28 

Number  of  cultures  acid,          -        0014  2     3     13     24  ] 

[In  litmus  milk  the  mannite -fermenting  bacilli  first  produce  a  slight  acidity 
(1-3  days)  and  ultimately  become  alkaline  (15  days).  Strong's  bacillus  is 
the  only  strain  which  forms  acid  and  clot  (Morgan).] 

Indol  production. — The  Shiga  bacillus  produces  no  indol  in  culture.  Bacilli 
of  the  Flexner  type  vary,  some  strains  produce  indol,  others  do  not.  [Accord- 
ing to  Morgan  the  vast  majority  of  the  mannite-fermenting  group  form  indol, 
some  more  freely  than  others.] 

Growth  in  arsenical  and  carbolic  broth. — All  dysentery  bacilli  grow  in  broth 
containing  carbolic  acid  (0'075  per  cent.)  or  arsenious  acid  (O'l  per  cent.). 

Growth  on  vaccinated  media. — On  agar  which  has  already  served  for  the 
growth  of  the  Shiga  bacillus  neither  the  Shiga  bacillus  nor  the  bacilli  of  the 
Flexner  type  will  grow  :  the  typhoid  bacillus  gives  a  very  poor  growth,  the 
colon  bacillus  on  the  other  hand  grows  abundantly. 

Similar  results  are  obtained  with  agar  which  has  served  for  the  growth  of 
bacilli  of  the  Flexner  type. 

On  media  which  have  served  for  the  growth  of  the  typhoid  or  colon  bacillus 
neither  the  Shiga  bacillus  nor  bacilli  of  the  Flexner  type  grow. 

2.  Vitality. 

The  dysentery  bacillus  is  a  somewhat  delicate  organism.  In  culture  it 
does  not  live  for  more  than  3  or  4  weeks  :  in  infected  stools  it  appears  to  be 
quickly  destroyed  by  the  other  micro-organisms  present,  and  especially  by 
the  action  of  the  colon  bacillus,  so  that  it  cannot  be  isolated  after  48  hours. 
Direct  sunlight  and  desiccation  rapidly  destroy  the  bacillus  :  it  is  killed  in 
less  than  an  hour  at  58°  C.  :  and  in  sterile  spring  water  at  20°  C.  it  does  not 
live  more  than  8-10  days.  In  water  containing  saprophytic  organisms  the 
larger  the  number  of  such  organisms  the  more  quickly  does  the  dysentery 
bacillus  disappear,  and  at  the  ordinary  temperature  it  cannot  be  recovered 
after  2-10  days  (Vincent)  ;  the  higher  the  temperature  also  the  more  rapidly 
does  the  organism  vanish  :  this  may  [partly]  explain  the  frequency  of 
epidemic  dysentery  in  cold  and  temperate  climates  (Vincent). 


BIOLOGICAL  PROPERTIES  361 


3.  Toxin. 

(i)  Filtered  cultures  of  the  dysentery  bacillus  are  generally  only  slightly 
toxic,  and  even  in  large  doses  merely  cause  a  temporary  loss  of  weight ;  but 
the  blood  of  the  inoculated  animal  acquires  the  property  of  agglutinating  the 
bacillus.  Todd  and  Rosenthal  however  obtained  a  strong  toxin,  which  was 
fatal  to  rabbits  in  doses  of  0'2  c.c.  sub-cutaneously,  by  growing  the  bacillus 
in  Martin's  broth  at  37°  C.  for  3  weeks  and  then  filtering.  Their  results  have 
been  confirmed  by  Ludke  and  Doerr.  , 

(ii)  Unfiltered  cultures  of  the  dysentery  bacillus  killed  by  heat  (58°  C.  for 
1  hour  or  85°  C.  for  30  minutes)  or  chloroform,  when  inoculated  into  rabbits 
intra-peritoneally,  intra-venously  or  sub-cutaneously,  lead  to  a  fatal  result 
similar  to  that  produced  by  the  living  organism  and  accompanied  by  diarrhoea 
and  hypersemia  of  the  mucous  membranes  of  the  colon  (Drigalski  and 
Conradi). 

(iii)  Endotoxin. — Conradi,  Neisser  and  Shiga,  Vaillard  and  Dopter,  and 
Besredka  have  extracted  an  intra-cellular  toxin  from  the  bodies  of  the 
bacilli. 

Conradi' s  method. — Scrape  the  growth  from  an  agar  culture  and  make  into  an 
emulsion  with  normal  saline  solution.  Place  the  emulsion  in  the  incubator  at  37°  C. 
and  leave  for  about  30  hours,  then  decant  the  clear  liquid,  filter  through  a  Berkefeld 
bougie  and  finally  evaporate  in  vacuo  to  one- tenth  its  original  volume. 

2.  Neisser  and  Shiga' s  method. — Heat  an  emulsion  of  bacilli  in  normal  saline 
solution  to  60°  C.,  allow  to  stand  for  48  hours  at  37°  C.  then  filter  through  a  Reichel 
filter.    • 

3.  Vaillard  and  Dopter' s  method. — Make  a  thick  emulsion  of  bacilli  from  an  agar 
culture  with  sterile  water,  heat  to  58°  C.  for  an  hour,  distribute  in  tubes,  seal  in  the 
blow-pipe  and  leave  in  the  warm  (37°  C.)  incubator  for  a  month.     The  clear  super- 
natant liquid  is  used  without  filtration. 

4.  Besredka' s  method. — Besredka  has  applied  his  method  of  extracting  endotoxin 
(p.  379)  to  the  dysentery  group.     The  endotoxin  is  very  toxic  and  kills  rabbits  in 
doses  of  0'05  c.c. 

The  Shiga  bacillus  alone  produces  dysentery  toxin,  bacilli  of  the  Flexner  type 
being  almost  invariably  atoxic. 

c/  U 

Properties  of  dysentery  toxin. — The  toxin  kills  dogs,  rabbits  and  mice  with 
all  the  symptoms  of  a  dysentery  infection.  The  fatal  dose  for  rabbits  varies 
according  to  the  method  of  preparation  and  the  method  of  inoculation  (intra- 
peritoneal.  intra-venous  or  sub-cutaneous)  from  O05  c.c.  (with  Besredka's 
endotoxin)  to  2-5  c.c.  Administered  by  the  mouth  it  gives  rise  to  no 
symptoms. 

Dysentery  toxin  is  less  affected  by  heat  than  are  many  other  toxins  :  it 
is,  for  instance,  unaltered  by  being  exposed  to  70°  C.  for  an  hour,  but  a  tem- 
perature of  75°  C.  weakens  it  and  at  80°  C.  its  properties  are  rapidly  destroyed. 
It  is  now  admitted  that  dysentery  toxin  is  not  a  soluble  or  diffusible  toxin  but 
an  endotoxin  retained  within  the  bodies  of  the  bacilli. 

4.  Vaccination.    Serum  therapy. 

(i)  Shiga  has  shown  that  animals  can  be  immunized  by  inoculating  them 
sub-cutaneously  with  small  doses  first  of  dead  bacilli  then  of  living  bacilli. 
The  serum  of  immunized  animals  agglutinates  the  bacillus  and.  has  both 
prophylactic  and  curative  properties. 

Small  animals  are  very  difficult  to  immunize  and  it  is  therefore  better  to 
use  a  goat  or  an  ass  or  an  horse  ;  horses  must  be  treated  very  carefully. 

The  serums  (ass  and  horse)  obtained  by  Shiga  and  by  Kruse  protect  guinea- 


362  THE   DYSENTERY   BACILLUS 

pigs  against  the  inoculation  of  a  fatal  dose  of  bacilli  and  have  powerful 
agglutinating  properties  (1-10,000).  Good  results  have  also  been  obtained 
in  the  treatment  of  human  dysentery  ;  with  Shiga's  serum  the  mortality  was 
reduced  two-thirds.  The  results  with  Martini  and  Lentz's  goat  serum  and 
with  Krauss  and  Doerr's  serum  (prepared  by  sub-cutaneous  inoculation  of 
living  cultures)  have  not  been  so  satisfactory. 

(ii)  Gay,  who  repeated  and  confirmed  Shiga  and  Kriise's  experiments, 
found  that  the  agglutinating  and  prophylactic  properties  of  dysentery- 
immunized  horse  serum  were  more  marked  with  the  strain  used  for  immuniza- 
tion than  with  other  strains.  Independently  of  this  Krauss  and  Doerr, 
thinking  that  their  experiments  showed  that  a  Shiga  immune  serum  had  no 
action  on  bacilli  of  the  Flexner  type  and  conversely,  recommended  the  pre- 
paration of  a  mixed  Shiga-Flexner  serum  which  could  be  given  indifferently 
in  all  cases  of  dysentery  whatever  the  infecting  organism  :  acting  on  this 
suggestion,  Coyne  and  Auche  prepared  a  polyvalent  serum  which  has  yielded 
satisfactory  results  in  the  treatment  of  dysentery  in  man.  Vaillard  and 
Dopter  however  affirm  that  a  Shiga-serum  gives  as  good  results  in  a  Flexner 
as  in  a  Shiga  infection,,  and  in  consequence  consider  that  polyvalent  serums 
are  unnecessary. 

(iii)  To  obtain  a  serum  which  was  both  anti-bacterial  and  antitoxic 
Rosenthal  immunized  horses  by  inoculating  them  subcutaneously  with 
toxin  (p.  361)  and  living  cultures  simultaneously.  The  serum  is  both  prophy- 
lactic and  curative  :  it  protects  guinea-pigs  in  doses  of  0*5  c.c.,  and  in  the 
treatment  of  human  dysentery  very  good  results  have  been  obtained,  especially 
by  Korentchewsky  in  Manchuria  where  the  mortality  from  dysentery  fell 
by  more  than  one-half. 

(iv)  Vaillard  and  Dopter,  relying  upon  an  observation  by  Besredka  to  the 
effect  that  in  the  case  of  endotoxic  organisms  the  most  active  antiserums  are 
obtained  when  the  bacilli  are  inoculated  directly  into  the  blood-stream,  im- 
munized horses  by  inoculating  living  cultures  and  toxin  directly  into  the 
veins. 

The  process  of  immunization  must  be  carried  out  very  slowly.  Virulent  cultures 
of  the  Shiga  bacillus  were  used  and  the  toxin  was  prepared  by  Rosenthal' s  method 
(p.  361).  Increasing  doses  of  cultures  were  inoculated  alternately  with  toxin,  com- 
mencing with  0'25  c.c.,  then  0'5.  1,  2,  3  c.c.,  rising  to  50  c.c.,  an  amount  which  was 
never  exceeded.  The  animals  reacted  violently  (fever,  prostration,  temporary 
paralysis  of  the  hind  limbs,  loss  of  weight).  The  serum  was  collected  a  fortnight 
to  three  weeks  after' the  last  inoculation. 

Vaillard  and  Dopter's  serum  is  both  anti-bacterial  and  antitoxic.  It  pro- 
tects rabbits  when  given  in  doses  of  0*25-0'5  c.c.,  and  leads  to  recovery 
(doses  of  1-2  c.c.)  even  when  administered  24  hours  after  the  experimental 
infection  In  doses  of  20-100  c.c.  it  is  very  efficient  in  the  treatment  of 
human  dysentery  :  the  symptoms  are  alleviated  almost  at  once,  recovery  is 
rapid  and  the  mortality  lowered  more  than  three-fourths. 

Vaccination  in  man. — Shiga  tried  to  vaccinate  men  by  inoculating  them  with  a 
mixture  of  killed  bacilli  (80  parts)  and  serum  (20  parts).  The  results  were  encourag- 
ing, but  the  immunity  is  only  of  short  duration. 

Immunity  can  be  quickly  produced  by  the  inoculation  of  serum  alone  but  does 
not  last  long  (10-12  days). 

In  future  experiments  it  would  seem  to  be  better  to  work  with  sensitized  bacilli 
according  to  the  technique  of  Besredka :  emulsions  of  bacilli  are  killed  by  heat 
and  agglutinated  by  a  non-heated  specific  serum  the  excess  of  serum  being  removed 
by  repeated  washing  and  centrifuging  (p.  382).  Such  a  vaccine  is  only  slightly  toxic  : 
in  mice  an  immunity  lasting  3-4  months  is  acquired  in  4  days,  and  the  susceptibility 
of  the  animals  is  not  increased  during  the  process  of  immunization. 


AGGLUTINATION  363 

5.   Agglutination. 

Shiga  was  the  first  to  show  that  the  serum  of  persons  suffering  from 
dysentery  agglutinates  the  bacillus. 

1.  Agglutinating  properties  of  experimental  serums. — The  serum  of  an 
immunized  animal  has  the  property  of  agglutinating  the  bacillus  used  for  its 
immunization.     The  most  highly  agglutinating  serums   (^^^•^i^^^)   are 
obtained  by  inoculating  animals  intra-venously. 

(a)  The  serum  of  normal  animals  has  no  action  on  the  dysentery  bacillus. 
((3)  Antidysentery  serum  is  specific  and  has  no  agglutinating   action  on 
the  typhoid,  colons  or  paratyphoid  bacilli. 

(7)  The  serum  of  animals  immunized  with  the  Shiga  bacillus  agglutinates 
that  organism  but  has  no  action  on  bacilli  of  the  Flexner  type. 

(8)  The  serum  of  animals  immunized  with  bacilli  of  the   Flexner  type 
agglutinates  these  bacilli  but  not  the  Shiga  bacillus. 

[(e)  Hiss'  Y  bacillus  is  agglutinated  by  a  Flexner  serum  but  not  by  a 
Shiga  serum.  Strong's  bacillus  is  agglutinated  neither  by  a  Shiga  nor  by  a 
Flexner  serum.] 

2.  Agglutinating    properties    of    the    serum    of   persons    suffering    from 
dysentery. — In  testing  the  agglutinating  properties  of  the  serum  of  a  dysentery 
patient  it  is  important  to  recognize  that  the  Shiga  bacillus  is  only  agglutinated 
in  low  dilutions  (rarely  in  dilutions  higher  than  -V~TJo)  an(i  that  bacilli  of 
the  Flexner  type  are  agglutinated  in  much  higher  dilutions  (5^)  and  may 
even  be  agglutinated  by  normal  serums  in  low  dilution. 

(a)  The  serums  of  healthy  persons  [exceptis  excipiendis]  and  of  patients 
suffering  from  diseases  other  than  dysentery  do  not  agglutinate  the  dysentery 
bacilli. 

(j3)  The  serum  of  patients  suffering  from  amoebic  dysentery  does  not 
agglutinate  the  dysentery  bacilli. 

(7)  The  serum  of  patients  suffering  from  bacillary  dysentery  agglutinates 
the  bacillus  causing  the  infection  and  with  rare  exceptions  has  no  action  on 
the  other  type  :  the  serum  of  a  patient  infected  with  the  Shiga  bacillus 
agglutinates  the  Shiga  bacillus  while  having  no  action  on  bacilli  of  the 
Flexner  type,  and  vice  versa. 

Whatever  the  type  of  bacillus  causing  the  infection  agglutination-capacity 
is  present  in  the  serum  only  in  severe  or  averagely  severe  cases  ;  it  seldom 
appears  before  the  end  of  the  first  week  and  occasionally  not  until  later,  but 
remains  for  several  weeks  after  recovery. 

(S)  The  serums  of  dysentery  patients  never  agglutinate  the  typhoid 
bacillus  ;  but  such  a  serum  may  agglutinate  some  varieties  of  the  colon 
bacillus,  the  explanation  being,  possibly,  that  a  colon  bacillus  infection  has 
been  superadded  upon  the  original  dysentery  infection. 

6.  Precipitins. 

If  one  drop  of  Shiga  serum  be  added  to  ten  drops  of  a  filtered  culture  of 
the  Shiga  bacillus  a  precipitate  is  formed  :  a  similar  but  less  marked  pre- 
cipitate is  also  formed  if  instead  of  the  Shiga  culture,  a  culture  of  one  of  the 
bacilli  of  the  Flexner  type  be  used. 

Conversely,  Flexner  serum  precipitates  filtered  cultures  of  bacilli  of  the 
Flexner  type  and  also  but  less  markedly  filtered  cultures  of  the  Shiga  bacillus. 

7.  Immune  body. 

In  the  serum  of  persons  suffering  from  dysentery  and  also  in  the  serum  of 
immunized  animals  a  specific  immune  body  is  present  which  is  fixed  both 


364  THE   DYSENTERY   BACILLUS 

by  the  bacillus  causing  the  infection  and  by  all  other  types  of  dysentery  bacilli. 
In  human  subjects  the  immune  body  makes  its  appearance  about  the  fifth  to 
the  seventh  day  of  the  disease.  It  is  quite  distinct  from  the  agglutinin  and 
may  be  present  in  the  serum  before  the  latter. 


SECTION  IV.— DETECTION,   ISOLATION  AND   IDENTIFICATION   OF 
THE   DYSENTERY  BACILLUS. 

To  ensure  the  detection  of  the  bacillus  in  a  case  of  dysentery  it  is  necessary 
to  examine  a  recently  evacuated  stool.  The  bacilli  are  most  numerous  in 
the  latter  during  the  first  week  of  the  disease,  but  subsequently  diminish 
in  number,  and  finally  disappear  altogether  as  soon  as  the  stools  resume 
their  normal  consistency.  Bacilli  cannot  be  found  in  the  stools  after  the 
twenty-first  day  of  the  disease  (Rosenthal). 

Dysentery  bacilli  may  occasionally  be  found  in  the  mesenteric  glands  and 
very  exceptionally  in  other  organs. 

The  bacilli  cannot  be  differentiated  by  microscopical  examination  alone 
and  cultures  must  be  sown  in  every  case. 

Select  a  flake  of  sero-sanguinolent  matter  and  after  washing  it  thoroughly 
in  sterile  water  emulsify  in  a  little  broth  ;  use  the  emulsion  for  sowing  gelatin 
or  agar  plates.  Plates  may  also  be  sown  by  smearing  the  surface  of  the 
medium  with  one  of  the  flakes  after  washing  it.  Should  there  be  no  mucous 
flakes  dilute  a  trace  of  the  stool  in  broth. 

(i)  Gelatin  plates  should  be  sown  by  the  dilution  method  (p.  78)  and  incu- 
bated at  22°  C.  After  2  or  3  days  the  surface  colonies  are  examined  and  any 
which  resemble  colonies  of  the  dysentery  bacillus  picked  off  for  further 
tests. 

(ii)  The  best  method  is  to  use  a  lactose-agar  medium — Chantemesse's(p.  407), 
Conradi-Drigalski's  (p.  407),  Endo's  (p.  408)  [or  M'Conkey's  (p.  412)]. 

Dip  a  fine  sterile  camel-hair  brush  in  the  broth  emulsion  and  smear  the  sur- 
face of  a  number  of  plates  of  this  medium  without  recharging  the  brush.  It  is 
perhaps  even  better  to  smear  the  surface  of  the  medium  with  a  washed  mucous 
flake  and  spread  the  material  with  a  Drigalski's  spatula  (p.  407).  Incubate 
at  37°  C.  When  examined  after  20-24  hours  colonies  of  the  colon  bacillus 
will  appear  as  red  spots,  while  those  of  the  dysentery  bacillus  and  of  some 
other  organisms  will  not  have  altered  the  colour  of  the  medium.  From 
among  the  latter  pick  off  those  which  have  a  translucent  iridescent  appear- 
ance with  irregular  margins  and  the  centres  of  which  are  rather  more  opaque 
than  the  edges,  and  sow  them  in  broth  and  other  media.  Dysentery  bacilli 
will  be  recognized  by  the  absence  of  motility,  by  the  cultural  characteristics 
mentioned  above  and  by  their  being  agglutinated  by  a  specific  serum.  [For 
purposes  of  identifying  bacilli  of  the  Flexner  type  the  serum  of  an  animal 
immunized  with  the  Y  bacillus  is  the  most  generally  useful  (Morgan).] 

Serum  diagnosis. 

Since  specific  agglutinins  are  present  in  the  serum  of  patients  suffering  from 
dysentery  it  is  possible  to  make  a  diagnosis  by  the  serum  reaction. 

Knowing  that  the  serum  of  patients  only  agglutinates  the  bacillus  which 
is  causing  the  infection  the  serum  must  be  "tested  both  with  a  Shiga  bacillus 
and  with  a  Flexner  bacillus.  The  reaction  towards  che  Shiga  bacillus  should 
be  tested  in  the  first  instance  with  a  dilution  of  1  in  20  or  1  in  30  ;  a  positive 
result  under  these  conditions  will  be  conclusive.  The  reaction  towards 
bacilli  of  the  Flexner  type  should  be  tested  in  a  dilution  of  1  in  80  ;  agglutina 


THE   EL  TOR  STRAIN  365 

tion  in  a  lower  dilution  cannot  be  accepted  as  evidence  of  a  Flexner  infection 
because,  as  has  already  been  pointed  out,  normal  serum  in  low  dilution  often 
has  an  agglutinating  action  on  this  type  of  the  bacillus. 

In  dysentery,  as  in  enteric  fever,  a  positive  reaction  confirms  the  diagnosis  : 
on  the  other  hand  if  no  reaction  be  obtained  bacillary  dysentery  cannot  be 
definitely  excluded,  because  the  blood  may  have  been  collected  before 
agglutinins  had  developed. 

[Bacillus  dysentericus  El  Tor  No.  I.1] 

This  organism  which  was  described  by  Armand  Ruffer  in  1909,  was  found  to 
be  the  cause  of  the  largest  percentage  of  the  cases  of  dysentery  among  the  Mussulman 
pilgrims  passing  through  the  lazaret  at  El  Tor :  it  has  the  characteristics  of  the 
dysentery  group,  but  appears  to  differ  from  all  the  known  sub-groups  at  present 
described.  Ruffer,  however,  remarks  that  the  name  is  merely  provisional  and  that 
the  bacillus  may  prove  to  be  identical  with  one  of  the  bacilli  already  described. 

Morphology. — The  bacillus  Tor  No.  1  is  similar  to,  but  plumper  than,  the  Shiga 
bacillus ;  filaments  were  rarely  seen ;  no  spores  were  found  nor  could  cilia  be 
demonstrated.  It  showed  movement  of  spiral  rotation  when  freshly  isolated  but 
no  movement  of  progression  :  in  sub-cultures  it  was  quite  motionless. 

Cultures. — Broth. — Uniform  turbidity  :    no  pellicle. 

Gelatin. — Not  liquefied.  The  colonies  have  no  vine-leaf  appearance  like  those  of 
the  Shiga  bacillus. 

Agar. — Similar  to  typhoid. 

On  Endo's  medium. — Colourless. 

On  Conradi-Drigalski's  agar. — Like  typhoid. 

Bio-chemical  reactions. —  B.  dysentericus  Tor  No.  1  formed  acid  out  of  mannite 
and  thus  resembled  Flexner' s  bacillus.  In  saccharose,  maltose,  salicin,  sorbite, 
dulcite  and  dextrin  reactions  were  very  inconstant  and  differed  with  different  strains 
of  the  bacillus. 

A  small  amount  of  indol  was  formed  sometimes  but  the  reaction  was  not  constant. 

In  milk,  no  clot  was  formed. 

In  litmus  milk  most  strains  produced  first  an  acid  reaction  followed  by  a  stage  of 
increased  alkalinity.  One  of  the  strains  however  produced  a  permanent  acidity. 

Pathogenicity. — The  B.  dysentericus  Tor  No.  1  was  highly  pathogenic  to  rabbits 
on  intra-peritoneal  or  intra- venous  inoculation,  giving  rise  to  fever,  diarrhoea  and 
paralysis  and  causing  death  in  24  hours  to  5  days  according  to  the  dose  inoculated. 
The  pathogenicity  for  rabbits  rapidly  disappeared  in  sub-cultures. 

Guinea-pigs  were  even  more  susceptible  than  rabbits.  The  symptoms  and  lesions 
were  the  same  as  in  the  rabbit. 

In  horses  inoculation  of  sterilized  cultures  produced  a  wide-spread  oedema  about 
the  site  of  inoculation,  rise  of  temperature  and  a  general  condition  of  ill-health. 

In  man  the  clinical  signs  and  pathological  appearances  were  indistinguishable 
from  those  produced  by  infection  with  the  Shiga  bacillus. 

Toxin. — None  of  the  cultures  of  this  organism  gave  a  soluble  toxin. 

Agglutination. — The  serum  of  patients  suffering  from  a  Tor  No.  1  infection  did 
not  agglutinate  the  Shiga  bacillus  but  agglutinated  bacillus  Tor  No.  1  constantly 
except  in  early  acute  cases  or  in  very  feeble  old  people.  The  dilution  in  which 
agglutination  could  be  obtained  varied  from  1  in  25  to  1  in  300,  the  index  rising 
during  convalescence. 

With  the  serum  of  animals  specifically  immunized  it  was  found  that  the  Tor 
bacillus  was  agglutinated  in  dilutions  of  1-1000  to  1-2000  with  a  Tor  serum,  while 
with  a  Shiga  serum  agglutination  was  inconstant  in  a  dilution  of  1-100,  and  with 
a  Flexner  serum  agglutination  was  effected  with  a  dilution  of  1—200  but  not  with  a 
dilution  of  1-500. 

Serum  therapy. — Ruffer  found  that  patients  suffering  from  a  Tor  infection  were 
not  benefited  by  treatment  with  a  Shiga  serum  while  severe  cases  were  quickly 
cured  by  inoculation  with  the  serum  of  an  horse  immunized  with  B.  dysentericus 
Tor  No.  1. 

[irThis  section  has  been  added.] 


CHAPTER  XXL 
BACILLUS  FEBRIS  ENTERICS. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  367. 

A.  Inoculation  of  viruses  of  ordinary  virulence,  p.  368.     B.  Inoculation  of  viruses 
of  exalted  virulence. — Methods  of  increasing  virulence. — Infection  with  viruses  of 
exalted  virulence,  p.  368.     C.  Infection  by  the  alimentary  canal,  p.  369. 
Section  II. — Morphology,  p.  370. 

1.  Microscopical     appearance     and     staining     reactions,     p.     370.      2    Cultura 
characteristics,  p.  371. 
Section  III. — Biological  properties,  p.  373. 

1.  Bio-chemical  reactions,  p.  373.     2.  Variability  of  flagella,  p.  376.     3.  Viability 
and  virulence,  p.  376.     4.  Toxins,  p.  376.    5.  Vaccination,  p.  380.    6.  Serum-therapy, 
p.  383.    7.  Agglutination  and  the  serum  diagnosis  of  enteric  fever,  p.  384.    8.  Absorp- 
tion of  agglutinins,  p.  389.     9.  Complement  fixation,  p.  390. 
Section  IV. — Detection,  isolation  and  identification  of  the  typhoid  bacillus,  p.  390. 

THE  causa]  organism  of  enteric  fever  was  originally  discovered  by  Eberth 
in  the  spleen,  lymphatic  glands  and  Peyer's  patches  of  persons  suffering  from 
the  disease.  Its  morphological  characteristics  were  more  fully  described  by 
Gaffky. 

The  bacillus  of  enteric  fever  1  is  always  present  in  the  spleen,  liver,  mesenteric 
glands,  glandular  follicles  of  the  intestine  and  bone  marrow  and  less  frequently  in 
the  lungs,  meninges,  testicles,  tonsils,  etc.  A  certain  number  of  cases  of  enteric 
fever  have  been  recorded  in  which  there  was  no  intestinal  localization. 

For  a  long  time  it  was  thought  that  the  bacillus  did  not  pass  into  the  blood  stream 
(Chantemesse  and  Widal,  and  others).  It  is,  however,  now  recognized  that  the 
failure  to  find  the  bacillus  in  the  blood  was  due  to  the  defective  technique  then 
employed  and  that,  as  a  matter  of  fact,  in  enteric  fever  the  bacillus  does  pass  into 
the  blood  stream  and  that  the  disease  is  in  reality  a  true  septicaemia.  In  all  cases  of 
moderate  and  severe  infection  the  organism  can  be  isolated  from  the  blood  from 
the  fifth  day  until  the  end  of  the  third  week  of  the  disease  (Courmont). 

The  bacillus  can  often  be  isolated  from  blood  taken  from  the  rose  spots  (Thiemisch 
and  Neuhaus,  Besson,  etc.). 

Remy's  experiments  have  proved  that,  contrary  to  the  opinion  formerly  held, 
the  bacillus  is  present  in  the  stools  of  enteric  fever  patients  as  early  as  the  third  day 
of  the  disease  and  before  ulceration  of  the  intestine  has  begun.  The  number  of 
organisms  present  in  the  stools  increases  until  the  end  of  the  first  week  and  then 
gradually  diminishes  until  at  the  end  of  the  fourth  week  they  can  as  a  rule  no  longer 
be  found.  The  bacilli  have,  however,  been  isolated  from  the  stools  of  persons  who 
have  recovered  from  the  disease  for  more  than  a  month  (Remy,  Chantemesse  and 
Decobert)  and  it  will  be  shown  later  that  in  some  cases  they  persist  for  a  still  longer 
period. 

1  In  the  remainder  of  the  chapter  and  elsewhere  the  organism  is  termed  for  the  sake 
of  convenience  the  typhoid  bacillus. 


INTRODUCTION  367 

The  bacillus  also  sometimes  passes  into  the  urine  of  enteric  fever  patients.  Besson 
from  an  examination  of  thirty-three  cases  came  to  the  conclusion  that  the  bacillus 
was  only  present  when  there  was  albumin  in  the  urine  and  found  it  in  40  per  cent, 
of  such  cases  :  the  bacillus  disappears  synchronously  with  the  disappearance  of  the 
albumin.  Vincent  found  the  bacillus  in  the  urine  in  about  1  case  in  5  of  enteric 
fever;  he  noticed  that  occasionally  the  bacillus  remained  in  the  urine  after  the 
patient  had  recovered,  and  considered  that  under  those  conditions  the  organism 
multiplied  in  the  bladder.  Horton  Smith  showed  that  the  bacillus  ~may  set  up 
slight  cystitis  with  pyuria. 

The  bacillus  is  also  the  cause  of  many  of  the  complications  of  enteric  fever,  such 
for  instance  as  inflammation  of  the  fauces,  naso-pharynx  and  larynx,  broncho- 
pneumonia  and  various  suppurative  affections :  deep  seated  abscesses,  osteitis, 
adenitis,  pleurisy,  pericarditis,  etc.  It  may  also  become  localized  in  lesions  existing 
before  the  onset  of  the  infection.  Widal  observed  instances  of  this  hi  a  case  of 
ovarian  cyst  and  in  a  case  of  tuberculous  adenitis. 

Chantemesse  was  the  first  to  put  forward  the  opinion  afterwards  supported 
by  Remlinger  and  Schneider  that  the  typhoid  bacillus  might  live  a  sapro- 
phytic  existence  in  the  intestines  of  healthy  persons.  The  investigations  of 
Remy  and  others,  however,  seem  to  prove  that  the  bacillus  is  only  found  as 
a  saprophyte  in  the  intestines  of  those  who  have  recently  been  either  in 
contact  with  cases  of  the  disease  or  in  some  other  way  exposed  to  infection. 
But  further,  in  a  certain  percentage  (according  to  Schneider,  3  per  cent.)  of 
patients  who  have  recovered  from  the  disease,  and  especially  in  women, 
the  bacillus  may  remain  for  several  months  and  even  years  :  it  is  said  to  take 
up  its  abode  principally  in  the  gall  bladder  from  whence  it  is  discharged  into 
the  intestine.  It  is  easy  to  appreciate  the  prominent  part  which  such 
"  carriers,"  to  use  Drigalski  and  Conradi's  expression,  may  take  in  the  dis- 
semination of  enteric  fever. 

The  bacillus  has  been  frequently  found  in  drinking  water  and  in  ice  destined 
for  human  consumption.  Wherever  enteric  fever  is  epidemic  the  drinking 
water  should  be  examined  for  the  presence  of  the  typhoid  bacillus. 

The  organism  has  also  been  isolated  from  soil  and  from  the  dust  of  wards 
in  which  cases  of  enteric  fever  have  been  nursed,  etc. 

The  attention  of  observers  has  been  drawn  to  the  part  which  flies  may  possibly 
play  in  the  propagation  of  the  disease.  During  an  epidemic  of  enteric  fever  at 
Chicago,  Mrs.  Hamilton  on  several  occasions  obtained  cultures  of  the  typhoid, 
bacillus  by  sowing  flies  which  had  been  caught  in  water-closets,  enteric  wards,  etc. 
Ficker  has  shown  that  flies  which  have  been  in  contact  with  cultures  of  the  typhoid 
bacillus  may  specifically  contaminate  objects  on  which  they  settle  even  as  long 
as  23  days  afterwards. 

The  typhoid  bacillus  and  the  colon  bacillus  are  in  many  ways  very  like  one 
another  and  both  have  their  usual  habitat  in  the  intestines  of  man  and  the 
lower  animals.  The  analogies  which  undoubtedly  exist  between  these  two 
organisms  have  led  some  observers  to  express  the  opinion  that  they  are 
identical.  This  view  however  has  not  met  with  general  acceptance  and  it  is 
now  clear  that  the  colon  bacillus  and  the  bacillus  of  enteric  fever  have  each 
their  own  characteristic  properties  and  are  in  fact  two  distinct  though  closely 
related  species 

SECTION  I.— EXPERIMENTAL   INOCULATION. 

The  lower  animals  are  not  naturally  susceptible  to  enteric  fever.  The 
inoculation  of  laboratory  cultures  is  in  most  cases  without  result ;  some 
observers  indeed  have  noticed  symptoms  of  intoxication  in  guinea-pigs, 
rabbits  and  mice,  but  they  have  not  been  able  to  produce  a  generalization  of 
the  bacillus.  If,  however,  a  virus  of  increased  virulence  be  inoculated  these 


368  THE  TYPHOID  BACILLUS 

animals  die  with  the  lesions  of  septicaemia.  In  monkeys  and  rabbits  typical 
attacks  of  enteric  fever  have  been  induced  by  feeding  with  typhoid  bacilli. 

A.  Inoculation  of  viruses  of  ordinary  virulence. — Even  cultures  which  have 
been  sown  with  material  direct  from  a  case  of  enteric  fever  do  not  as  a  rule 
lead  to  a  generalized  infection  in  the  lower  animals,  though  occasionally 
guinea-pigs  and  mice  can  be  infected  by  inoculating  them  in  the  peritoneal 
cavity.     Sub-cutaneous  inoculation  generally  results  in  the  formation  of  a 
small  abscess  at  the  site  of  inoculation  from  which  the  animal  rapidly  recovers. 

In  rabbits,  guinea-pigs  and  dogs,  intra-cranial  inoculation  of  a  small  amount 
(O05-O1  c.c.)  of  a  fifteen-  or  twenty-day  old  culture  gives  rise,  by  reason  of 
the  toxin  it  contains,  to  severe  symptoms  which  terminate  fatally.  The 
inoculation  of  young  cultures  produces  nothing  more  than  a  transitory 
illness  (Vincent). 

B.  Inoculation  of  viruses  of  exalted  virulence. — Sanarelli,  Chantemesse  and 
Widal  and  others  have  succeeded  in  increasing  the  virulence  of  typhoid  bacilli 
and  with  these  exalted  viruses  they  can  always  be  certain  of  producing  a 
typhoid  septicaemia  in  laboratory  animals. 

Methods  of  increasing  virulence.— (a)  Sanarelli  inoculated  5  c.c.  of  a 
twenty-four-hour  old  broth  culture  of  a  typhoid  bacillus  of  ordinary  virulence 
into  the  cellular  tissue  of  a  guinea-pig  and  at  the  same  time  into  the  peritoneal 
cavity  10  c.c.  of  an  old  sterilized  broth  culture  of  the  colon  bacillus  :  death 
supervened  in  about  20  hours  and  post  mortem  the  typhoid  bacillus  was  found 
in  the  peritoneal  cavity  and  occasionally  also  in  the  spleen  and  blood. 

A  little  of  the  peritoneal  exudate  from  this  animal  was  then  sown  on  broth 
and  it  was  found  that  5  c.c.  of  the  broth  culture  sub-cutaneously  inoculated 
into  a  second  guinea-pig  would  kill  the  animal  if,  at  the  same  time,  7-8  c.c. 
of  a  sterilized  culture  of  the  colon  bacillus  were  inoculated  intra-peritoneally. 
By  thus  passing  the  bacillus  through  a  series  of  animals  diminishing  at  each 
inoculation  the  dose  of  colon  bacillus  culture,  it  happened  that  after  a  short 
time  a  strain  of  the  typhoid  bacillus  was  recovered  which  could,  unaided  by 
the  simultaneous  inoculation  of  the  colon  bacillus,  lead  to  an  enteric  infection 
in  rabbits  and  guinea-pigs  when  inoculated  sub-cutaneously  in  doses  of  5  c.c. 

•  Similar  results  were  obtained  by  Sanarelli  if  instead  of  the  colon  bacillus  he 
inoculated  sterilized  cultures  of  Proteus  vulgaris,  sterilized  cultures  of  stools,  or  an 
infusion  of  meat  a  month  old  sterilized  at  120°  C.  By  simply  feeding  guinea-pigs 
with  small  quantities  of  this  infusion  he  was  able  to  secure  the  generalization  of  a 
typhoid  bacillus  which  before  had  no  pathogenicity  for  the  guinea-pig. 

In  the  case  of  a  virus  which  is  fatal  to  guinea-pigs  in  large  doses  the  viru- 
lence may  be  raised  by  passage  intra-peritoneally  through  guinea-pigs.  For 
this  purpose  2  or  3  c.c.  of  a  peritoneal  exudate  rich  in  bacilli  are  inoculated 
in  the  first  instance,  then,  as  the  virulence  increases,  as  evidenced  by  the  fact 
that  the  animals  die  in  a  shorter  space  of  time  and  by  the  diminished  quantity 
of  exudate  found  post  mortem,  the  quantity  injected  is  gradually  reduced  to 
0-5  and  O'l  c.c.  After  fifteen  to  twenty  such  passages  a  single  drop  is  suffi- 
cient to  kill  an  adult  guinea-pig  in  12  hours.  After  the  thirtieth  passage  the 
virulence  is  fixed  and  cannot  be  further  increased.  A  few  drops  of  a  twenty- 
four-hour  old  broth  culture  of  the  "  fixed  virus  "  is  sufficient  to  kill  susceptible 
animals  on  intra-peritoneal  inoculation.  If  inoculated  sub-cutaneously 
much  larger  doses  must,  however,  be  employed  :  thus,  for  instance,  in  the 
case  of  rabbits  and  guinea-pigs  1-4  c.c.  and  for  mice  0'5  c.c.  are  necessary. 

Note. — In  attempting  to  raise  the  virulence  of  an  organism  by  passage  through  the 
peritoneal  cavities  of  guinea-pigs  it  is  important  to  utilize  the  peritoneal  exudate 
itself  for  the  successive  inoculations  and  not  cultures  sown  from  the  exudates.  To 


EXPERIMENTAL   INOCULATION  369 

maintain  the  virulence  after  exaltation  the  organism  should  be  grown  on  a  broth 
which  before  sterilization  turns  phenol- phthalein  pink  and  to  which  a  few 
drops  of  guinea-pig  blood  have  been  added  just  before  sowing  it  (Rodet  and 
Lagriffoul). 

(b)  ChaDtemesse  and  Widal  also  raised  the  virulence  of  bacilli  moderately 
virulent  [for  experimental  animals]  by  passage  through  guinea-pigs,  utilizing 
to  that  end  a  discovery  of  Vincent  relative  to  the  exaltation  of  the  typhoid 
bacillus  when  associated  with   sterile  cultures  of  streptococcus  pyogenes. 
They  inoculated  into  the  cellular  tissues  of  a  guinea-pig  4  c.c.  of  a  culture 
of  a  typhoid  bacillus  and  at  the  same  time  into  the  peritoneal  cavity  8-10  c.c. 
of  a  culture  of  a  pyogenic  streptococcus  which  had  been  sterilized  at  100°  C. 
for  1  hour.     The  animal  died  in  less  than  24  hours  and  the  typhoid  bacillus 
was  found  to  have  become  generalized.     The  organism  was  passed  through  a 
series  of  guinea-pigs  and  the  dose  of  sterilized  streptococcus  emulsion  gradu- 
ally diminished,  with  the  result  that  the  typhoid  bacillus  soon  became  so 
virulent  that  a  few  drops  introduced  into  the  peritoneal  cavity  caused  the 
death  of  the  animal. 

(c)  According  to  Chantemesse  and  Balthazard  the  most  efficient  method  of 
raising  the  virulence  of  a  typhoid  bacillus  to  a  maximum  is  to  sow  a  culture 
in  a  collodion  sac,  and  after  leaving  it  in  the  peritoneal  cavity  of  a  guinea- 
pig  for  24-36  hours  to  sow  the  Contents  in  broth  :  the  growth  is  very  abundant 
so  that  in  12  hours  the  surface  is  covered  with  a  thick  pellicle.     This  culture 
is  fully  virulent. 

Infection  with  viruses  of  exalted  virulence. — Guinea-pigs  are  the  best 
animals  for  the  study  of  typhoid  infections.  A  few  drops  of  an  exalted  virus 
inoculated  into  the  peritoneal  cavity  gives  rise  to  a  typical  attack  of  the 
disease. 

Two  to  four  hours  after  inoculation  the  temperature  rises  and  may  reach 
41°  C.  but  it  soon  (6-12  hours)  begins  to  fall  to  36°  C.  and  perhaps  32°  C.  ; 
synchronously  with  the  fall  of  temperature  collapse  sets  in  and  the  animal 
dies  15-30  hours  after  the  inoculation. 

During  the  febrile  period  the  animal  is  dull  and  refuses  its  food.  When 
the  temperature  has  become  subnormal  it  huddles  itself  up  in  a  corner  of  its 
cage,  the  abdomen  is  painful  and  the  animal  rapidly  wastes. 

Post  mortem  examination.  The  peritoneal  cavity  is  found  to  contain  a 
variable  amount  of  an  opalescent  serous  fluid  very  rich  in  bacilli  (the  greater 
the  virulence  of  the  organism  the  less  the  effusion)  :  the  spleen,  liver,  kidneys, 
intestines  and  notably  the  Peyer's  patches  are  swollen  and  congested  :  the 
mesenteric  glands  are  swollen  and  in  some  cases  there  is  a  little  pleural  effu- 
sion :  the  -intestine  contains  a  serous  fluid  rich  in  bacilli.  According  to 
Chantemesse  and  Widal  these  latter  are  typhoid  bacilli  but  according  to 
Sanarelli  they  are  very  virulent  colon  bacilli. 

The  organism  is  found  in  pure  culture  in  the  peritoneal  exudate  and  also 
in  the  internal  organs,  blood  etc. 

C.  Infection  by  the  alimentary  canal.  1.  Monkeys. — Chantemesse  and 
Ramond  fed  a  Macacus  rhesus  for  a  fortnight  on  an  exclusively  milk  diet 
and  then  gave  it  a  virulent  agar  culture  of  the  typhoid  bacillus  mixed  with 
jam.  As  early  as  the  third  day  the  animal  experienced  a  rise  of  temperature, 
anorexia  and  diarrhoea,  and  was  dead  at  the  end  of  a  week.  Post  mortem 
examination  revealed  lesions  characteristic  of  human  enteric  fever  especially 
in  the  neighbourhood  of  Peyer's  patches. 

2.  Rabbits. — Kemlinger  succeeded  in  infecting  rabbits  by  starving  them  for 
2  or  3  days  and  then  feeding  them  for  5-10  days  on  vegetables  contaminated 
with  cultures  of  the  typhoid  bacillus.  Many  of  the  animals  remained 

2A 


370  THE   TYPHOID   BACILLUS 

unaffected  but  a  few  of  them  towards  the  end  of  the  first  week  had  a  rise  of 
temperature,  became  emaciated,  suffered  from  diarrhoea  and  eventually 
died.  Post  mortem,  examination  showed  ulceration  of  Peyer's  patches, 
enlargement  of  the  spleen,  etc.  The  typhoid  bacillus  was  recovered  in  pure 
culture  from  the  spleen. 

Chantemesse  and  Ramond  lowered  the  resistance  of  rabbits  by  injecting 
into  the  peritoneal  cavity  some  sterile  broth  containing  50  drops  of  laudanum 
and  then  a  quarter-of-an-hour  later  introduced  into  the  stomach  by  means 
of  a  tube  5  c.c.  of  a  young  broth  culture  of  the  typhoid  bacillus.  Animals 
so  treated  became  infected  with  a  true  enteric  fever  ;  they  developed  the 
characteristic  lesions,  and  their  serum  agglutinated  the  bacillus. 

By  daily  inoculation  with  human  blood  serum  or  urine  for  a  period  of 
3  weeks  animals  can  be  rendered  more  susceptible  to  infection  with  the 
typhoid  bacillus. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  typhoid  bacillus  occurs  in  the  tissues  as  a  short  rod  measuring  about 
2-3/x  long  and  0'6-0'7/x  broad. 

In  cultures  its  length  and  breadth  vary  within  wide  limits.  In  broth,  for  example, 
the  bacillus  is  shorter  and  more  slender  ;  in  old  gelatin  cultures,  it  is  elongated  and 
shows  filamentous  forms ;  on  agar  and  potato  it  is  broader  and  shorter  and  has 
a  squat  appearance. 

The  bacilli  both  in  tissues  and  cultures  occur  singly  or  joined  together  in 
pairs,  and  in  young  cultures  they  not  infrequently  look  like  diplococci. 

The  ends  of  the  bacilli  are  rounded. 
The  protoplasm  stains  uniformly,  but 
occasionally,  in  old  cultures,  the  bacilli  are 
somewhat  swollen  about  their  centres  and 
show  a  clear  space  of  variable  size — "  the 
shuttle  form  "  of  Artaud.  This  unstained 
portion  does  not  represent  spore  formation 
any  more  than  do  the  terminal  swellings 
which  are  sometimes  seen  in  cultures  of 
the  bacillus  and  which  are  merely  de- 
generation forms. 

As  a  rule,  the  typhoid  bacillus  is  very 
motile  and  moves  rapidly  across  the  field 
of  the  microscope  like  fish  in  water,  but 
some  strains  of  the  bacillus  are  only  slightly 
FIG.  216.— Film  preparation  of  the  typhoid    motile.      The  motility  is  due  to  the  pre- 
bacillus    from   a   gelatin   culture.     Carbol-  f  a        „      /    .  /   •    /•     N 

fuchsin.   (Oc.  2,  obj.  /2th,  Zeiss.)  sence  ol  nagella  (vide  infra). 

If  a  trace  of  growth  from  a  solid  medium 

be  placed  in  a  drop  of  water  the  bacilli  separate  one  from  another  and  the 
water  is  immediately  rendered  turbid  (Chantemesse). 

Staining  reactions.— The  typhoid  bacillus  stains  readily  with  the  basic 
aniline  dyes  and  is  gram-negative. 

Staining  of  flagella.— The  nagella  may  be  easily  stained  (p.  149).  Van 
Ermengem  s  or  Nicolle's  method  is  recommended  as  giving  the  best  results. 

In  stained  films  the  number  and  arrangement  of  the  flagella  can  be  readily 
made  out.  As  a  rule,  each  bacillus  has  eight  to  a  dozen  flagella,  but  it  is  not 
at  all  uncommon  for  individual  bacilli  to  have  as  many  as  eighteen  to 
twenty-four.  Flagella  which  have  been  inadvertently  torn  away  from 


MORPHOLOGY 


371 


their  bacilli  during  the  necessary  manipulations  will   be  found  in  every 
preparation. 

The  flagella  are  normally  implanted  regularly  around  the  body  of  the 
organism  [peritrichous  ]  though  now  and  again  they  are  found  arranged  in 
tufts  probably  from  the  dragging  of  the  surround- 
ing liquid  on  these  highly  delicate  structures. 
The  bacilli  are  often  agglutinated  into  clumps 
by  a  matrix  which  stains  in  the  same  manner 
as  the  flagella  and  it  is  upon  this  matrix  that 
the  flagella  appear  to  be  implanted. 

The  flagella  vary  in  length,  the  average  being 
(Remy  and  Sugg) ;  but  much  longer  forms 
are  to  be  seen.  They  are  wavy  in  form  and 
present  three  to  eight  undulations. 

2.  Cultural  characteristics. 

A.  Conditionsofsrowth.-ThetyphoidbaciUus 

is  a  facultative  aerobe.     It  grows  on  all  the 

ordinary  media  within  a  wide  range  of  temperature  (4°-46°  C.)  the  optimum 

being  30°-37°  C.     Cultures  of  the  typhoid  bacillus  have  no  smell. 

B.  Characters  of  growth  on  various  media.    1.  Broth. — After  8-12  hours' 
incubation  at  37°  C.  the  medium  shows  a  slight  cloudiness,  which  as  the  growth 
progresses  becomes  more  marked,  and  gives  to  the  culture  when  examined 
by  transmitted  light  a  characteristic  watered-silk  appearance  :  this  may  be 
made  more  distinct  by  gently  shaking  the  tube  :    later  the  growth  becomes 
flocculent,  falls  to  the  bottom  of  the  tube,  and  forms  a  very  abundant  sedi- 
ment.    Ultimately  the  liquid  becomes  clear  and  develops  a  brownish  colour. 

2.  Gelatin. — The  typhoid  bacillus  does  not  liquefy  gelatin. 
Stab  culture. — At  20°  C.  growth  along  the  line  of  the  stab  commences  as 
early  as  the  second  day  in  the  form  of  small,  round,  yellowish-white  confluent 
colonies,  while  on  the  surface  a  thin,  transparent,  rather  spread-out  disc 
with  iridescent  margins  appears  ;    occasionally  the  surface  growth  is  repre- 
sented by  a  thick   opaque   spot   of 
very  limited  extent.    Growth  is  always 
scanty. 

Stroke  culture.  —  On  the  surface 
along  the  line  of  sowing  the  growth 
forms  a  thin  transparent  film  with 
irregular  margins  and  shot  with  iri- 
descent colours  ;  it  always  remains 
scanty  and  ceases  to  increase  after 
the  first  week.  Such  is  the  usual 
appearance,  but  sometimes  a  narrow, 
thick,  opaque,  yellowish- white  band 
develops  along  the  stroke. 

In  the  substance  of  the  gelatin 
long  arborescent  crystals  are  some- 
times seen.  These  are  due  to  the 
precipitation  of  phosphates. 

Single  colonies. — Isolated  colonies 
on  gelatin  usually  but  not  invariably 
present  a  characteristic  appearance.  After  incubating  at  20°  C.  for  48  hours, 
small  circular  colonies  appear  and  soon  reach  the  size  of  a  pin's  head  and 
later  that  of  a  lentil,  but  always  remain  thin,  bluish  in  colour,  pearly  and 


FIG.  218. — Typhoid  bacillus.  Photograph  of  a  colony 
growing  in  plate  culture  (6  days),      x  60. 


372 


THE  TYPHOID  BACILLUS 


FIG.  219.— Typhoid  bacillus. 
Culture  on  potato. 


transparent :  the  edges  of  each  colony  become  indented  and  sinuous,  and  at 
the  same  time  ridges  extend  from  them  into  the  centre,  which  becomes 
thicker  than  the  margins.  These  details  may  be  made  out  clearly  with  a 
lens.  The  general  appearance  has  been  compared  by  German  writers  to  an 
iceberg. 

Colonies  developing  in  the  depth  of  the  gelatin  and  sometimes  even  those 
on  the  surface  have  quite  a  different  appearance.  They  are  round  and 
opaque,  and  remain  about  the  size  of  a  pin's  head. 

3.  Agar  :    Coagulated  serum. — There  is  nothing  characteristic  about  the 
growth  on  these  media.     After  incubating  for  24  hours  at  37°  C.  a  whitish 
streak  appears,  which  subsequently  becomes  thicker  and  cream-coloured. 
Glycerin-agar  yields  a  more  copious  growth. 

4.  Potato. — The  growth  of  the  typhoid  bacillus  on  potato  is  as  a  rule 
characteristic.     At  first  sight  there  appears  to  be  no  growth  at  all :    but  on 

illuminating  the  surface  of  the  potato  by  day- 
light a  delicate,  moist,  shiny  deposit  like  the 
icing  on  cakes  is  seen  along  the  line  of  sowing. 
Sometimes  the  culture  assumes  a  bistre  tint 
later. 

In  some  cases  however  the  growth  on  potato 
is  plainly  visible,  being  yellowish  in  colour  and 
occasionally  even  definitely  brownish.  Buchner 
states  that  this  appearance  can  be  obtained  at 
will  by  making  the  potato  alkaline  with  a  solution 
of  carbonate  of  soda. 

5.  Remy  and  Sugg's  medium. — To  avoid  complications  induced  by  varia- 
tions in  the  chemical  composition  of  potato,  an  artificial  medium  has  been 
prepared  by  Kemy  and  Sugg  which  contains  the  constituent  ingredients  of 
potato.     According  to  the  authors  the  typhoid  bacillus  on  this  medium 
invariably  gives  a  characteristic  growth  ;    "  a  limited,  absolutely  colourless, 
scalloped  film." 

The  medium  is  prepared  as  follows. 
(a)  Make  a  solution  containing  : — 

Water,     - 

Glucose,  - 

Peptone, 

Asparagin, 

Citric  acid, 

Neutral  potassium  phosphate, 

Magnesium  sulphate, 

Potassium  sulphate,  - 

Sodium  chloride, 

Carbonate  of  sodium  q.s.  to  render  the  whole  slightly  alkaline. 
(6)  To  100  c.c.  of  this  solution  add  :— 

Gelatin  (extra  quality), 10  grams. 

Calcined  magnesia,    ---.....  2 

Distribute  in  tubes,  sterilize,  slope.     Sow  in  stroke  culture. 

8l  B^er~Sterilized  ox-bile  is  a  very  useful  medium  on  which  to  grow  the 
typhoid  bacillus  (Conradi).  It  is  used  as  an  "  enrichment  medium  "  for 
obtaining  [primary]  cultures  from  material  in  which  the  bacillus  is  only 
present  in  small  numbers,  as  for  example  the  blood  of  enteric  fever  patients 

7.  Milk.— The  bacillus  grows  abundantly  in  milk  without  coagulating  the 
medium. 


1000  c.c. 
20  grams. 
5 
5 

0-75  gram. 
5  grams. 
2-5      „ 
2-5     „ 

1-25  ,: 


BIOLOGICAL  PROPERTIES  373 


SECTION  III.— BIOLOGICAL  PROPERTIES. 

The  difficulty  of  distinguishing  the  typhoid  from  the  colon  bacillus  has 
rendered  necessary  a  close  study  of  the  biological  properties  of  the  two 
organisms :  the  morphological  characteristics  alone  are  insufficient  to  allow 
of  their  differentiation. 

1.  Biochemical  reactions.1 

Action  on  carbohydrates. — The  typhoid  bacillus  has  a  distinct  action 
[acid  without  apparent  gas]  upon  glucose,  [maltose,  sorbite  and  mannite] 
and  also  acts  feebly  upon  Isevulose  and  galactose,  but  ferments  neither 
saccharose,  lactose,  [dulcite,  raffinose,  arabinose,  erythrite,  salicin,  amygdalin 
nor  inulinj. 

These  properties  furnish  valuable  data  for  the  recognition  of  the  organism, 
and  the  methods  of  demonstrating  them  will  now  be  considered. 

(a)  Sow  the  bacillus  in  a  tube  of  lactose-broth  to  which  a  little  carbonate 
of  lime  has  been  added  (p.  35).  No  gas  is  formed  however  long  the  culture 
be  incubated. 

(6)  Sow  on  litmus-lactose-gelatin  (p.  57)  :  the  typhoid  bacillus  does  not 
attack  either  mannite  or  lactose  so  that  no  acid  is  formed  and  the  medium 
retains  its  blue  colour  (cf.  Bacillus  coli). 

(c)  Sow  in  Grimbert  and  Legros'  medium.     This  medium  has  the  following 
composition  : — 

Lactose  (chemically  pure),  20  grams. 

Peptone,  5         ,, 

Distilled  water,  -       1000  c.c. 

Dissolve  by  boiling  :  add  a  little  pure  carbonate  of  lime :  shake  :  leave  for 
5  minutes  :  filter  :  test  the  reaction,  which  should  be  neutral.  Sterilize  by  filtering 
through  a  Chamberland  bougie.  Distribute  into  tubes  and  add  sufficient  sterilized 
litmus  solution  (p.  56). 

After  sowing  with  the  typhoid  bacillus  and  incubating,  the  medium  retains 
its  blue  colour. 

(d]  Sow  in  milk. — The  milk  is  not  coagulated  and  if  a  little  litmus  solution 
be  added  its  colour  remains  unchanged. 

[A  definite  acidity  is  produced  in  the  first  24  hours  but  this  is  subsequently 
neutralized  and  the  medium  ultimately  becomes  distinctly  alkaline,  though 
the  time  occupied  in  the  production  of  an  alkaline  reaction  varies  considerably 
with  different  strains — in  some  cases  a  month  may  elapse  before  the  medium 
is  definitely  alkaline.  ] 

For  these  tests  the  milk  should  always  be  sterilized  at  the  same  known  tempera- 
ture :  some  contaminating  organisms  which  easily  coagulate  milk  which  has  been 
sterilized  at  100°  C.  coagulate  it  more  slowly  and  with  more  difficulty  if  it  has  been 
exposed  to  higher  temperatures,  and  mistaken  diagnosis  may  result  if  this  fact  has 
not  been  recognized  (see  also  p.  57). 

These  reactions  are  sufficient  to  enable  the  typhoid  bacillus  to  be  dis- 
tinguished from  the  colon  bacillus  (p.  393).  When  it  is  necessary  to  make  a 
differential  diagnosis  between  the  typhoid  and  colon  bacilli  glucose  should 
never  be  used  as  the  fermentable  agent  since  the  typhoid  bacillus  has  a  distinct 
action  on  it. 

Non-production  of  indol. — The  typhoid  bacillus  never  produces  indol  in 
cultures. 

1  Here  the  nature  of  the  reactions  will  be  briefly  stated ;  their  application  to  the 
differentiation  of  the  typhoid  and  colon  bacilli  will  form  the  subject  of  a  special  chapter 
(xxiii.). 


374  THE   TYPHOID   BACILLUS 

Tests  for  indol. — To  determine  whether  an  organism  produces  indol  or  not, 
a  solution  of  peptone  must  be  used  and  not  ordinary  broth.  The  following 
is  a  medium  often  used  for  this  test : — 

Water,     -  -         100  c.c. 

Witte's,  Chapoteaut's  or  Byla's  peptone,  2  grams. 

Sodium  chloride,       -  0*5  to  1  gram. 

Tube  in  quantities  of  about  15  c.c.  and  autoclave. 

After  sowing,  incubate  for  2-8  days  and  apply  one  or  other  of  the  following 
tests : — 

(a)  Salkowski's  reaction. — To  the  culture  in  peptone  water  add  1  c.c.  of  a 
0'2  per  cent,  solution  of  potassium  nitrite,  then,  slowly,  1  c.c.  of  a  25  per 
cent,  solution  of  chemically  pure  sulphuric  acid  in  water.  If  indol  be  present 
a  rose  tint  appears. 

Nonotte  and  Demanche  find  that  the  reaction  is  more  delicate  in  the  warm.  To  a 
peptone-water  culture  add  1  c.c.  of  a  1  in  1000  solution  of  nitrite  of  potassium  and 
8  drops  of  pure  concentrated  sulphuric  acid  and  boil  the  upper  part  of  the  liquid. 
If  indol  be  present  a  very  distinct  pink  colour  appears  even  when  the  amount  of 
indol  does  not  exceed  1  part  in  4  millions  :  in  the  cold,  the  reaction  only  takes  place 
if  the  amount  of  indol  exceeds  1  part,  in  75,000. 

(6)  Weyl-Legal's  reaction. — To  the  culture  add  5  to  10  drops  of  a  5  per 
cent,  solution  of  sodium  nitro-prusside  then  a  few  drops  of  a  30  per  cent, 
solution  of  washing  soda.  The  solution  turns  brown.  After  a  few  minutes 
add  10  to  15  drops  of  glacial  acetic  acid  ;  if  indol  be  present  a  characteristic 
blue  colour  appears  but  often  only  after  some  delay. 

(c)  Nencki's  reaction. — To  the  culture  add  first  a  few  drops  of  glacial  acetic 
acid  then  2-3  c.c.  of  alcohol-ether  :   shake  and  allow  to  stand  until  the  ether 
rises  :  decant  the  layer  of  ether  and  evaporate  it  in  a  porcelain  dish.     To  the 
residue  add  1  to  2  drops  of  a  0'2  per  cent,  solution  of  potassium  nitrite  and  a 
few  drops  of  pure  sulphuric  acid.     This  method  is  very  delicate  and  the  least 
trace  of  indol  is  shown  by  the  appearance  of  a  rose  pink  colour. 

(d)  Fleig's  reaction.— To  10  c.c.  of  culture  add  10  c.c.  of  a  1  in  50  alcoholic 
solution  of  furfurol,  then  pure  hydrochloric  acid  drop  by  drop.     If  indol  be 
present  the  solution  turns  yellow.     This  method  is  very  delicate. 

[(e)  The  para-dimethyl-amido-benzaldehyde  test.    Recommended. — Prepare 
two  solutions : 
Solution  I. — 

Para-dimethyl-amido-benzaldehyde,   -  4  parts. 

Absolute  alcohol,       -  -         380      ;. 

Concentrated  hydrochloric  acid,  -         -         -         -  80       „ 

Solution  II. — 

Saturated  aqueous  solution  of  potassium  persulphate. 

[To  about  10  c.c.  of  the  broth  or  peptone-water  culture  of  the  organism 
add  5  c.c.  of  Solution  I.  and  then  5  c.c.  of  Solution  II.,  shake  the  mixture  and 
the  presence  of  indol  is  indicated  by  the  appearance,  in  a  very  short  time,  of 
a  red  colour,  which  gradually  becomes  darker  on  standing.  The  reaction 
may  be  accelerated  by  heating  the  mixture.  | 

Some  peptones  contain  a  trace  of  indol  and  to  avoid  all  possibility  of  mistake 
Sicre  recommends  using  a  1  per  cent,  solution  of  Byla's  peptone  and  when  testing 
for  indol  to  test  at  the  same  time  a  tube  of  sterilized  peptone- water  as  a  control. 

Growth  on  Synthetic  media.— A  number  of  synthetic  media  have  been 
prepared  on  which  the  typhoid  bacillus  grows  slowly  and  feebly  while  closely 
related  organisms  with  which  it  may  be  confused  grow  freely. 

Too  much  importance  should  not  be  attached  to  the  differentiating  function  of 
these  media,  but,  generally  speaking,  if  growth  be  absent  or  delayed  on  any  one  of 


BIOLOGICAL  PROPERTIES  375 

them,  this  is  an  indication  sufficiently  reliable  to  justify  a  suspicion  of  the  presence 
of  the  typhoid  bacillus. 

For  choice,  the  following  medium,  composed  by  Remy  and  Sugg,  may  be  used  : — 
Distilled  water,  -          -          -          -          -  1000  c.c. 


Glucose, 

Nitrate  of  soda, 

Magnesium  sulphate, 

Neutral  phosphate  of  potassium, 

Calcium  chloride, 


20  grams. 

10  „ 
2  „ 
1  gram. 


Inability  to  grow  on  "  vaccinated  "  media. — Chantemesse  and  Widal  have 
demonstrated  the  following  curious  property  of  the  typhoid  bacillus.  If 
a  tube  of  agar  or  gelatin  be  sown  with  the  bacillus  and  after  incubation 
the  growth  be  scraped  off  and  the  medium  resown  with  the  organism  the 
second  sowing  remains  unfertile  on  incubation,  the  medium  having  been,  as 
it  were,  "  vaccinated  "  by  the  first  growth.  Unfortunately  this  phenomenon 
sometimes  fails  and  taken  alone  is  not  a  reliable  test.  The  colon  bacillus 
also  frequently  fails  to  grow  on  a  medium  which  has  been  used  for  the  growth 
of  the  typhoid  bacillus. 

Growth  on  coloured  media. — D'Abundo  ;  Nceggerath ;  and  also  Gasser  have 
drawn  attention  to  the  property  possessed  by  the  typhoid  bacillus  of 
decolourizing  during  growth  media  stained  with  certain  dyes. 

Nceggerath's  medium  (p.  57)  was  recommended  by  its  discoverer  and  later  by 
Deschamps  and  Grancher  as  a  diagnostic  agent  for  the  typhoid  bacillus.  When 
sown  on  the  surface  of  gelatin  plates  coloured  with  Nceggerath' s  fluid,  the  typhoid 
bacillus  gives  rise  to  colonies  of  a  purple  colour  while  the  surrounding  medium 
becomes  decolourized. 

Gasser,  recognizing  that  Nceggerath' s  medium  gives  inconstant  results,  substituted 
fuchsin-agar  (p.  57).  The  typhoid  bacillus  sown  on  this  medium  and  incubated  at 
37°-39°  C.  for  2  days  gives  red  colonies,  the  surrounding  medium  being  decolourized. 

These  reactions  are  unfortunately  not  constant  and  cannot  be  relied  upon 
for  the  purpose  of  recognizing  the  typhoid  bacillus. 

Growth  on  arsenical  broth. — Thionot  and  Brouardel  found  that  the  typhoid 
bacillus  does  not  grow  in  broth  containing  arsenious  acid  to  the  extent  of 
0'02  gram  per  litre,  while  the  colon  bacillus  grows  not  only  in  this  medium 
but  also  when  the  broth  contains  as  much  as  1-2  grams  of  arsenious  acid 
per  litre. 

Growth  on  artichoke. — According  to  Roget,  the  typhoid  bacillus  produces 
no  apparent  growth  on  artichoke  and  does  not  change  the  colour  of  the 
medium,  while  the  colon  bacillus  gives  a  thick  yellowish  growth  and  the 
artichoke  at  the  same  times  assumes  an  intense  green  colour. 

Technique. — Remove  the  leaves  of  the  artichoke  but  leave  the  choke  adhering  to 
the  heart :  cut  into  small  cubes  with  a  silver- bladed  knife,  place  the  cubes  with  the 
choke  uppermost  into  potato  tubes  containing  a  few  drops  of  water  in  the  lower 
bulb,  plug  with  wool  and  sterilize  at  115°  C.  Sow  at  the  junction  of  the  choke 
and  heart. 

Growth  on  caffeine  media. — Roth  has  shown  that  the  addition  of  0*5  per 
cent,  of  caffeine  to  media  prevents  the  growth  of  the  colon  bacillus  but  has 
no  action  on  the  growth  of  the  typhoid  bacillus.  This  characteristic  is  not 
absolutely  constant  since  some  strains  of  the  typhoid  bacillus  will  not  grow 
in  the  presence  of  caffeine  (Courmont). 

Growth  on  malachite-green. — According  to  Lceffler  the  addition  of  a  small 
amount  (about  1  in  4000)  of  malachite-green  to  culture  media  favours  the 
growth  of  the  typhoid  and  paratyphoid  bacilli  while  impeding  the  growth  of 


376  THE   TYPHOID   BACILLUS 

the  colon  bacillus.  Kiralyfi  has  shown  that  this  is  not  a  constant  pheno- 
menon :  according  to  this  observer  though  malachite-green  inhibits  the 
growth  of  many  micro-organisms — e.g.  streptococci,  staphylococci,  vibrio 
cholerse, — it  generally  has  no  action  on  the  colon  bacillus. 

Krystal-violet  has  the  same  action  as  malachite-green  (Drigalski  and  Con- 
radi) :  both  typhoid  and  colon  bacilli  grow  on  media  containing  this  dye  while 
the  growth  of  many  other  organisms  is  inhibited. 

2.  Variability  of  flagella. 

Sunlight,  antiseptics  in  dilute  solution  and  temperatures  unsuitable  to 
growth  have  practically  no  influence  on  the  number  and  shape  of  the  flagella 
(Kemy  and  Sugg).  When  the  typhoid  bacillus  has  been  grown  in  culture 
with  the  colon  bacillus  for  some  weeks  the  flagella  are  sometimes  difficult  to 
stain  (Kemy).  The  rarity  of  variation  in  the  morphology  of  the  flagella 
is  of  importance  in  diagnosing  the  typhoid  bacillus. 

3.  Viability  and  virulence. 

Viability.— Exposure  to  a  temperature  of  60°  C.  kills  the  typhoid  bacillus 
in  10-20  minutes  but  very  low  temperatures  have  no  effect  on  its  vitality  ; 
thus,  Prudden  found  the  organism  still  alive  in  a  block  of  ice  which  had  been 
kept  for  3  months  between  -  1°  and  -  11°  C.  On  the  other  hand,  alternate 
freezing  and  thawing  rapidly  kills  the  bacillus. 

In  water,  the  typhoid  bacillus  retains  its  vitality  for  some  time  (Strauss 
and  Dubarry,  Chantemesse  and  Widal).  In  sterile  water  it  has  been  found 
to  be  alive  after  3  months.  If  the  water  contains  saprophytic  micro-organisms 
the  typhoid  bacillus  disappears  more  quickly,  but  it  can  still  be  isolated  after 
more  than  1  month. 

In  soil,  the  bacillus  can  survive  five  months  and  a  half  (Grancher  and 
Deschamps)  :  drying  kills  it  only  after  1  or  2  months  (UfFelmann).  Levy 
and  Kaiser  isolated  the  organism  from  stools  which  after  being  in  a  cesspool 
for  5  months  had  been  spread  on  the  surface  of  the  ground  for  15  days  in 
winter. 

Light  rapidly  kills  the  typhoid  bacillus.  Cultures  exposed  to  sunlight  in 
the  month  of  May  were  sterilized  in  4—8  hours.  Vincent  has  found  that  the 
blue,  violet  and  ultra-violet  rays  are  more  efficient  as  bactericidal  agents 
than  the  red  and  ultra-red  rays.  Cultures  spread  and  dried  on  pieces  of 
cloth,  and  then  exposed  to  direct  sunlight,  were  found  to  be  sterilized  in 
9-26  hours  (Vincent). 

The  typhoid  bacillus  is  very  sensitive  to  the  action  of  antiseptics  :  the 
solutions  of  perchloride  of  mercury,  carbolic  acid,  etc.  in  general  use  will 
kill  the  bacillus  in  a  few  minutes. 

Virulence. — The  great  variations  observed  in  the  virulence  of  the  typhoid 
bacillus  and  the  methods  by  which  the  virulence  can  be  raised  have  already 
been  studied  under  the  head  of  experimental  inoculation. 

4.  The  toxin  of  the  typhoid  bacillus. 

The  experiments  conducted  by  Brieger  and  Fraenkel  with  a  view  to  isolating 
a  toxin  from  the  typhoid  bacillus  gave  very  little  result.  Attempts  are  now 
no  longer  made  to  extract  a  definite  chemical  substance  from  cultures.  The 
crude  toxin  found  in  sterilized  cultures  has  been  studied  by  Sanarelli,  Chante- 
messe and  others.  Other  observers  (Macfadyen  and  Rowland,  Besredka  and 
others)  have  prepared  extracts  containing  an  endotoxin  from  the  bodies  of 
the  organisms, 


TYPHOID   TOXIN  377 

1.  Toxin  of  Sanarelli.  (a)  Method  of  preparation. — Sanarelli  uses  a  virus 
the  virulence  of  which  has  been  raised  by  passing  it  through  the  peritoneal 
cavities  of  guinea-pigs  (p.  368).  The  bacillus  is  sown  in  2  per  cent,  glycerin- 
broth  and  after  incubating  at  37°  C.  for  a  month  is  sterilized  by  heat  and 
allowed  to  remain  at  room  temperature  for  8  months.  The  flask  containing 
the  culture  is  then  sealed  in  the  flame  and  heated  to  60°  C.  for  a  few  days. 
During  this  long  period  of  maceration  the  intra-cellular  toxin  diffuses  into 
the  culture  fluid  and  this,  carefully  decanted,  constitutes  the  toxin  of 
Sanarelli. 

Gauthier  and  Balthazard  justifiably  point  out  that  Sanarelli' s  toxin  is  a  complex 
mixture  containing  substances  foreign  to  the  typhoid  bacillus,  and  derived  partly 
from  the  albuminoid  substances  present  in  the  medium,  partly  from  the  dead  bodies 
of  the  bacilli,  etc.  which  have  slowly  undergone  disintegration.  It  is  nevertheless 
true  that  animals  inoculated  with  the  different  toxins  prepared  by  SanarelH,  Chante- 
messe,  and  Balthazard  exhibit  identical  symptoms. 

(/?)  Action  on  laboratory  animals.  On  rabbits. — The  toxin  given  sub- 
cutaneously  in  doses  of  10  c.c.  per  kg.  of  body  weight  kills  rabbits  weighing 
700-1000  grams. 

Soon  after  inoculation  the  animal  is  seen  to  breathe  more  rapidly  and  to  become 
unsteady  on  its  legs  ;  a  general  paralysis  gradually  comes  on  and  about  10  hours 
after  inoculation  convulsions  occur  ending  in  death.  The  temperature  is  at  first 
a  little  raised  (about  rV  C.)  but  soon  falls  below  normal  and  death  takes  place  while 
the  temperature  is  still  sub-normal.  The  effects  of  the  toxin  vary  in  different 
animals  :  death  is  not  infrequently  delayed  for  some  days  and  in  that  case  is  preceded 
by  a  period  of  cachexia  of  which  the  characteristic  signs  are  wasting,  diarrhoea,  etc. 
Post  mortem,  the  abdominal  organs  are  found  to  be  anaemic,  and  it  is  noticeable  that 
there  is  neither  congestion  of  the  intestinal  mucous  membrane  nor  swelling  of  the 
Peyer's  patches. 

On  mice. — 1  c.c.  of  toxin  inoculated  sub-cutaneously  or  0P2  c.c.  intra- 
peritoneally  is  generally  fatal,  death  taking  place  in  a  few  hours.  Post  mortem 
the  spleen  is  enlarged  and  there  is  a  small  amount  of  a  sterile  effusion  in  the 
peritoneal  cavity. 

On  guinea-pigs. — Guinea-pig  inoculation  is  an  excellent  means  of  testing 
typhoid  toxin — sub-cutaneously  the  minimal  fatal  dose  is  1'5  c.c.  per  100 

frams   of  body   weight.     Intra-peritoneally   the   results   are   less   constant, 
ub-cutaneous  inoculation  of  4  or  5  c.c.  of  toxin  per  100  grams  of  body  weight 
leads  to  death  in  15-20  hours. 

Prom  the  moment  of  inoculation  the  temperature  falls  and  continues  to  do  so  until 
death.  About  an  hour  after  inoculation  there  is  marked  abdominal  distension 
accompanied  by  extreme  tenderness,  the  animal  does  not  move,  but  sits  huddled  up 
and  cries  if  touched  :  after  4  or  5  hours  it  is  extremely  dejected,  its  eyes  are  half- 
closed  and  it  is  seen  to  be  in  an  almost  uninterrupted  state  of  tremor ;  a  profuse 
sometimes  haemorrhagic  diarrhoea  is  often  present,  and  finally  paralysis  appears, 
the  meteorism  vanishes  and  death  takes  place.  Post  mortem  a  variable  quantity 
of  exudate  rich  in  leucocytes  and  sometimes  turbid  is  found  in  the  peritoneal  cavity ; 
the  spleen  is  enlarged,  congested  and  friable  :  the  walls  of  the  small  intestine 
are  distended  and  completely  infiltrated  with  blood,  the  mucous  membrane  is  red 
and  the  lymphatic  patches  are  infiltrated  and  congested ;  the  stomach  and 
suprarenal  capsules  are  intensely  congested  and  ecchymosed.  The  intestine  is  full 
of  liquid  matter  and  contains  a  very  virulent  colon  bacillus  in  pure  culture. 

On  monkeys. — Monkeys  are  very  susceptible  to  the  toxin  of  the  typhoid 
bacillus  :  the  course  of  the  disease  and  the  lesions  are  the  same  as  in  the 
guinea-pig. 

2.  Toxin  of  Chantemesse.  (a)  Method  of  preparation. — Chantemesse  at 
first  recommended  growing  an  organism  of  increased  virulence  in  a  maceration 
of  spleen  and  bone  marrow.  He  now,  however,  prefers  to  use  a  bacillus 


378  THE   TYPHOID   BACILLUS 

whose  virulence  has  been  increased  by  growing  it  in  collodion  sacs  in  the 
peritoneal  cavities  of  guinea-pigs,  and  to  sow  it  in  a  solution  of  spleen  peptone.1 

Incubate  at  37°  C.  :  a  week  later  the  toxicity  will  be  at  a  maximum.  Then 
either  filter  through  porcelain,  or  preferably,  heat  to  55°  C.,  centrifuge,  and 
decant  the  supernatant  liquid  which  contains  the  toxin. 

(P)  Properties. — This  toxin  is  more  powerful  than  Sanarelli's  and  kills  a 
guinea-pig  weighing  500  grams  in  12-24  hours  when  inoculated  in  quantities 
of  6  c.c.  intra-peritoneally  (that  is  about  1  c.c.  per  80  grams). 

It  is  a  very  unstable  product,  being  quickly  affected  by  air  and  light,  and 
its  toxic  properties  are  diminished  if  it  be  heated  to  100°  C.  for  a  few  minutes. 
It  must  be  stored  in  accurately  filled  tubes  and  kept  in  the  dark. 

3.  Toxin  of  Bandi. — The  bacillus  after  the  virulence  has  been  raised  by 
passing  it  through  the  peritoneal  cavities  of  a  long  series  of  guinea-pigs  is 
sown  in  Lceffier's  broth,  incubated  for  4.8  hours  and  filtered.     The  filtrate 
inoculated  sub-cutaneously  in  quantities  of  4  c.c.  is  sufficient  to  kill  a  guinea- 
pig  weighing  400-500  grams. 

4.  Toxin  of  Le'pine  and  Lyonnet.— A  virulent  culture  in  broth,  4-8  days 
old,  is  sterilized  at  55°-60°  C.  for  an  hour.     The  product  is  toxic  for  dogs 
and  horses. 

5.  Toxin  of  Rodet,  Lagriffoul  and  Wanly.— Cultures  of  the  typhoid  bacillus 
are  incubated  on  well  aerated  media  for  3  days  and  filtered.     The  filtrate 
kills  guinea-pigs  when  inoculated  intra-peritoneally  in  doses  of  4  c.c.  per 
100  grams  of  body  weight,  and  rabbits  when  inoculated  intra-venously  in 
doses  of  1  c.c.  per  100  grams. 

M.  and  Mme.*  Werner  after  growing  the  organism  for  3  days  in  an  aerated  medium 
sealed  the  flasks  and  left  them  for  2  days  at  25°  C.  The  filtered  liquid  killed  guinea- 
pigs  when  inoculated  intra-peritoneally  in  quantities  of  £  c.c.  per  100  grams  of  body 
weight,  and  rabbits  when  inoculated  intra-venously  in  quantities  of  O'l  c.c.  per  100 
grams. 

6.  Toxin  of  Moreschi. — Moreschi  grew  the  bacillus  for  5  days  in  a  special 
broth  and  then  filtered  the  culture  through  porcelain.     The  filtrate  when 
injected  intra-peritoneally  in  doses  of  0'2  c.c.  killed  a  guinea-pig  weighing 
250  grams. 

The  special  broth  is  prepared  as  follows : — 

Mince  1000  grams  of  horse  meat  and  1000  grams  of  ox's  spleen,  macerate  for  24 
hours  at  room  temperature  in  a  litre  of  water,  boil,  filter,  make  up  to  1  litre  and  add 
Witte's  peptone,        -  20  grams. 

Plasmon,  ........  10         „ 

Sodium  chloride,       -         -         -         .          .         .          .          .  5         „ 

Ox  blood,         -         -  80 

Heat  the  mixture  to  120°  C.  in  the  autoclave  for  20  minutes,  neutralize,  and  add 
0-15  per  cent,  of  caustic  soda.  Heat  again  to  120°  C.  Filter,  tube  and  sterilize. 

After  being  sub- cultured  several  times  on  this  medium  the  bacillus  grows  as  a 
very  thick  film  on  the  surface  while  the  broth  remains  clear.  When  the  growth 
assumes  these  characteristics  the  culture  has  reached  its  maximum  of  toxicity. 

7.  Toxin  of  Gonradi. — The  bacillus  is  grown  on  agar  for  20  hours,  scraped 
off,  mixed  with  a  little  normal  saline  solution  and  kept  in  the  incubator  at 
37°  C.  for  24  hours.     The  emulsion  is  then  diluted  with  more  normal  saline 
solution  and  filtered  through  a  Berkefeld  bougie  ;   the  filtrate  is  evaporated 

1  The  medium  used  by  Chantemesse  is  prepared  by  macerating  spleen  and  bone  marrow 
in  cold  distilled  water,  filtering  through  porcelain  and  adding  a  little  defibrinated  human 
blood. 

The  solution  of  spleen  peptone  is  obtained  by  digesting  a  pig's  spleen  and  stomach  in 
acidulated  water  (vide  Martin's  peptone)  making  slightly  alkaline  and  sterilizing.  Cultures 
are  grown  in  a  shallow  layer  of  the  medium  contained  in  large  wide-bottomed  flasks. 


TYPHOID  TOXIN  379 

in  vacua  to  TVth  or  -f^fh  its  original  volume.  The  product  when  injected 
intra-peritoneally  in  doses  of  O2  c.c.  kills  a  guinea-pig  weighing  300 
grams. 

8.  Toxin  of  Macfadyen  and  Rowland. — The  growth  on  agar  is  scraped  off 
and  cooled  to  -  90°  C.  by  means  of  liquid  air,  then  triturated  at  a  very  low 
temperature  in  a  special  apparatus.     The  product  is  diluted  in  normal  saline 
solution  and  centrifuged.     The  supernatant  liquid  is  very  toxic  and  is  fatal 
to  guinea-pigs  when  inoculated  intra-peritoneally  in  doses  of  O'l  c.c. 

Bassenge  and  Mayer  obtained  a  similar  but  less  toxic  product  by  freezing 
the  bacilli  with  liquid  air  and  grinding  them  up  in  a  hand  mortar. 

9.  Toxin  of  Balthazard.     (a)  Mode  of  preparation.— The  principle  is  the 
same  as  that  underlying  Macfadyen  and  Rowland's  method. 

A  bacillus  whose  virulence  has  been  increased  by  growing  it  in  collodion  sacs  in 
the  peritoneal  cavities  of  guinea-pigs,  is  sown  on  large  surfaces  of  agar  contained  in 
flat  flasks  (the  agar  is  prepared  with  a  3  per  cent,  solution  of  Defresne's  peptone 
and  contains  no  meat).  After  incubating  for  24-48  hours  the  growth  is  scraped  off, 
mixed  with  a  little  normal  saline  solution  and  rapidly  centrifuged.  The  deposit 
is  again  shaken  up  with  normal  saline  solution  and  centrifuged  a  second  time.  In 
this  manner  all  foreign  matter  is  removed. 

The  bacilli  are  then  mixed  with  a  2  per  cent,  solution  of  urea  or  1  per  cent,  solution 
of  ammonium  chloride  (the  effect  of  these  solutions  is  to  swell  and  break  the  cells 
and  so  facilitate  the  diffusion  of  the  intra- cellular  products).  The  emulsion  thus 
obtained  is  distributed  into  tubes  which  are  completely  filled  and  sealed  in  the 
flame. 

To  facilitate  the  diffusion  of  the  intra-cellular  products,  the  tubes  are  now  alter- 
nately frozen  and  thawed.  They  are  kept  at  58°  C.  for  8  days  and  daily  submitted 
to  a  temperature  of  -21°  C.  for  a  couple  of  hours  in  a  refrigerating  machine  (the 
evaporation  of  methyl  chloride  being  adopted  as  the  cooling  agent).  At  the  end 
of  8  days  the  tubes  are  centrifuged  for  24  hours.  The  bacilli  collect  at  the  lower 
end  of  the  centrifuge  tube  and  the  supernatant  liquid  carefully  decanted  constitutes 
the  toxin. 

(/?)  Properties. — Balthazard's  method  though  lengthy  and  expensive 
yields  a  very  powerful  toxin,  containing  products  of  the  typhoid  bacillus 
unmixed  with  foreign  substances.  Inoculated  sub-cutaneously  in  doses  of 
3  c.c.  it  kills  rabbits  weighing  2  kg.  and  in  doses  of  2  c.c.  guinea-pigs  weighing 
200  grams.  It  is  however  less  toxic  than  Conradi's  toxin,  which  has  the 
further  advantage  of  being  more  easily  prepared. 

The  action  of  Balthazard's  toxin  on  animals  generally  is  similar  to  that 
of  the  toxins  prepared  by  Sanarelli  and  by  Chantemesse  ;  but  its  action  on 
rabbits  appears  to  be  more  constant. 

10.  Endotoxin  of  Besredka. — Dried  typhoid  bacilli  killed  by  heating  for  1 
hour  at  60°  C.  are  ground  up  with  sodium  chloride  until  an  impalpable  powder 
is  obtained.     This  powder  is  diluted  with  water  added  drop  by  drop  and  the 
mixture  left  over-night.     Next  morning  it  is  warmed  in  a  water  bath  to 
60°-62°  C.  for  2  hours,  and  then  allowed  to  settle.     The  supernatant  liquid 
contains  the  endotoxin.     The  average  lethal  dose  for  white  mice  is  0'05  c.c. 
intra-peritoneally.     The  endotoxin  is  destroyed  only  by  temperatures  above 
127°C. 

11.  Typho-lysin. — As  early  as  the  second  day  filtered  cultures  of  the  typhoid 
bacillus  show  distinct  powers  of  haemolysis.     This  property  increases  with 
the  age  of  the  culture  up  to  the  fifteenth  day  when  it  is  at  its  maximum 
(E.  and  P.  Levy).     Macfadyen   and   Rowland   demonstrated   the  presence 
of  typho-lysin  in  an  eight-day  culture  grown  on  macerated  spleen.     This 
haemolysin  of  the  typhoid  bacillus  is  not  destroyed  at  55°  C. 

The  red  cells  of  the  dog  are  very  sensitive  to  typho-lysin,  and  dogs  which  have 
been  repeatedly  treated  with  heated  cultures  yield  an  antitypholytic  serum. 


380  THE   TYPHOID   BACILLUS 

5.  Vaccination. 

A.  Immunization  of  the  lower  animals. 

(i)  Beumer  and  Peipper  immunized  white  mice  by  inoculating  them  daily 
for  several  days  with  increasing  doses  of  living  cultures.  Guinea-pigs,  rabbits, 
and  especially  goats  and  dogs  may  be  vaccinated  in  a  similar  manner  (Pfeiffer, 
Lceffler  and  Abel).  Vincent  immunized  dogs  and  rabbits  by  inoculating  them 
first  with  cultures  heated  to  60°  C.,  then  with  living  cultures  16  hours  old, 
and  finally  with  more  toxic  cultures  15-20  days  old.  The  serum  of  animals 
so  treated  has  both  immunizing  and  agglutinating  properties  :  immunized 
animals  are  however  not  immune  to  an  mtra-cerebral  inoculation  of  typhoid 
toxin. 

(ii)  Brieger,  Wassermann  and  Kitasato  used  for  their  immunizing  experi- 
ments organisms  attenuated  by  being  grown  in  thymus  broth  (p.  34). 
Inoculations  of  a  culture  of  a  virulent  bacillus  grown  in  thymus  broth  and 
heated  to  60°  C.  produced  immunity  in  guinea-pigs  and  mice. 

(iii)  Sanarelli,  Chantemesse  and  Widal,  Beumer  and  Peipper  immunized 
animals  by  inoculating  them  with  cultures  sterilized  by  heat. 

(a)  Sanarelli  incubated  a  culture  of  a  bacillus  of  increased  virulence  in 
peptone  broth  for  a  week  at  37°  C.  and  sterilized  the  growth  at  120°  C.  '  The 
sterilized  product  possessed  vaccinating  properties. 

Generally,  it  may  be  said  that  to  immunize  guinea-pigs  weighing  400  grams  it  is 
only  necessary  to  inoculate  them  several  times  over  a  period  of  5  days  with  16-18  c.c. 
of  sterilized  cultures.  The  animals  are  immune  4  days  after  the  last  inoculation 
and  will  then  resist  the  inoculation  of  a  virus  of  exalted  virulence.  During  the 
process  of  immunization  the  animals  lose  a  certain  amount  of  weight  but  quickly 
recover. 

It  is  very  difficult  to  immunize  rabbits  for  they  are  far  more  susceptible  than 
guinea-pigs  and  death  often  takes  place  during  the  immunizing  process  ;  but  animals 
which  survive  the  treatment  are  immune  in  a  high  degree. 

(6)  Chantemesse  and  Widal  used  broth  cultures  incubated  at  37°  C.  for 
15  days  and  then  sterilized  at  100°  C. 

Twenty  c.c.  are  necessary  to  immunize  a  guinea-pig  :  the  toxin  should  be  inocu- 
lated in  four  doses  allowing  a  few  days  to  elapse  between  each  inoculation. 
Immunization  takes  a  fortnight ;  after  the  lapse  of  another  8  days  the  test  inocula- 
tion may  be  performed  (2  c.c.  of  a  virulent  culture  into  the  peritoneum).  Not 
infrequently  the  animals  die  either  during  the  immunizing  process  or  as  the  result 
of  the  test  inoculation. 

Rabbits  may  be  immunized  in  a  similar  manner  but  in  these  animals  the  process 
is  even  more  difficult. 

(c)  Beumer  and  Pfeiffer  immunized  sheep  in  a  like  manner  with  cultures 
heated  to  60°  C.  for  an  hour. 

For  immunizing  horses  Funck  prefers  to  use  cultures  sterilized  with  carbolic 
acid. 

(iv)  Chantemesse  immunized  horses  by  injecting  them  with  gradually 
increasing  doses  of  his  toxin  (vide  supra). 

The  immunization  of  horses  is  very  difficult ;  the  inoculations  whether  made 
sub-cutaneously  or  intra-venously  have  frequently  to  be  interrupted  on  account 
of  the  violence  of  the  reaction,  and  it  takes  several  years  to  produce  a  lasting 
immunity. 

B.  Human  vaccination. 

For  many  years  the  problem  of  the  vaccination  of  the  human  subject 
against  enteric  fever  has  been  under  investigation.  In  1896,  Pfeiffer  and 
Kolle  showed  that  as  a  result  of  inoculating  man  with  a  small  quantity  of 


VACCINATION  381 

a    sterilized   culture *    the    serum   acquired   bactericidal   and   agglutinating 
properties  for  the  typhoid  bacillus. 

Since  then  numerous  methods  of  antityphoid  inoculation  have  been  devised, 
some  based  on  the  inoculation  of  "  whole  "  cultures  (Pfeiffer  and  Kolle, 
Wright,  etc.)  others  on  the  use  of  extracts  made  from  the  bodies  of  the  bacilli. 
Finally,  Besredka  has  conceived  a  method  of  vaccinating  with  bacilli 
sensitized  with  antityphoid  serum. 

1.  Methods  based  on  the  use  of  "whole"  cultures. 

(a)  The  Wright-Leishman  method.— Wright's  original  method  has  been 
modified  in  view  of  the  experiments  of  Leishman  and  Harrison.  Wright 
now  uses  a  bacillus  of  low  virulence  which  he  grows  at  37°  C.  for  24-48  hours 
in  a  shallow  layer  of  peptone  broth  to  facilitate  aeration,  sterilizes  by  heating 
to  53°  C.  for  an  hour  (not  at  60°  C.  as  in  his  original  method),  and  then  adds 
O25  per  cent,  of  lysol  to  ensure  its  sterility. 

Two  inoculations  into  the  outer  surface  of  the  arm  or  over  the  pectoral 
muscle  are  given  :  the  first  of  500  million  bacilli  (0'5  c.c.  of  vaccine),  the 
second  10  days  later  of  a  1000  million  bacilli  (1  c.c.  of  vaccine). 

The  doses  prescribed  by  Wright  and  Leishman  should  be  scrupulously  observed  : 
too  small  a  dose  will  fail  to  produce  immunity  and  too  large  a  dose  will  be  followed 
by  a  sharp  reaction  and  may  fail  to  vaccinate  (Wright,  Paladino-Blandini).  It  is 
therefore  necessary  to  enumerate  the  bacillary  content  of  the  vaccine  in  order  to 
standardize  it. 

Standardization  of  the  vaccine. — Mix  a  measured  volume  of  vaccine  with  an 
equal  volume  of  a  known  dilution  of  blood,  make  a  film,  stain  and  count  the  number 
of  bacilli  and  red  cells  in  several  fields  of  the  microscope.  The  number  of  red  cells 
per  cubic  centimetre  being  known,  the  number  of  bacilli  is  easily  calculated. 

(/3)  Pfeiffer  and  Kolle 's  method. — Cultures  on  agar  24  hours  old  are  scraped 
with  a  platinum  needle  and  the  growth  mixed  with  saline  solution  (45  c.c.  for 
ten  tubes).  The  emulsion  is  filtered  through  gauze,  the  filtrate  is  heated  to 
60°  C.  for  2  hours,  then  distributed  in  tubes  and  a  little  carbolic  acid  added. 
The  quantity  to  be  used  for  the  first  dose  is  0'5  c.c.  (corresponding  to  1  loopful 
or  2  milligrams  of  fresh  culture  or  y^th  of  an  agar  culture)  :  8  or  12  days 
later  a  second  dose  of  1  c.c.  is  given.  A  third  dose  may  with  advantage  be 
given  ;  if  this  be  purposed  it  is  well  in  order  to  obviate  any  violent  reaction 
to  use  smaller  doses  viz.  :  0'3  c.c.,  0'8  c.c.,  and  1  c.c. 

(7)  Bassenge  and  Rimpau's  method. — These  authors  adopt  a  technique 
similar  to  that  of  Pfeiffer  and  Kolle,  but  to  avoid  too  violent  a  reaction 
they  give  four  inoculations  of  very  small  quantities  with  an  interval  of  10 
days  between  each  :  for  the  first  inoculation  a  dose  equal  to  ^th  of  a  loopful 
is  given  and  then  successive  doses  of  y-th,  Jth,  and  ith  of  a  loopful. 

(S)  Friedberger  and  Moresehi's  method.— A  minimal  quantity  (T^th  or 
ToVoth  of  a  loopful)  of  an  eighteen-hour  culture  on  agar  dried  and  heated  to 
120°  C.  for  2  hours  is  inoculated  intra-venously.  A  single  inoculation  is 
sufficient  but  the  intra-venous  does  not  seem  as  harmless  as  the  sub-cutaneous 
method  and  is  followed  by  a  violent  reaction. 

2.  Methods  based  upon  the  use  of  bacillary  extracts. 

The  active  principle  of  the  typhoid  bacillus  can  be  extracted  by  the  different 
methods  which  have  been  studied  under  the  head  of  typhoid  toxin  :  macera- 
tion, trituration,  freezing,  etc.  Methods  of  antityphoid  vaccination  based 

1  At  first  it  was  the  custom  to  use  very  virulent  bacilli.  Wassermann  has  shown  that 
there  is  no  direct  and  constant  relation  between  toxigenic  and  immunizing  power  :  he 
suggests  the  use  of  a  polyvalent  vaccine  prepared  with  a  mixture  of  many  strains  of 
typhoid  bacilli ;  such  a  vaccine  is  said  however  to  have  no  advantage  over  a  monovalent 
vaccine  (Bassenge  and  Mayer). 


382  THE   TYPHOID   BACILLUS 

on  the  use  of  bacillary  extracts  are  complicated  and  do  not  seem  to  offer 
any  particular  advantages  over  those  just  considered. 

(a)  Wassermann's  method. — An  emulsion  of  cultures  on  agar  is  made  in 
distilled  water,  heated  to  60°  C.  for  24  hours,  macerated  for  5  days  at  37°  C., 
filtered  through  porcelain  and  dried  in  varuo  at  35°  C.  A  single  inoculation 
is  given  consisting  of  0*0017  gram  of  the  powder. 

(/3)  Neisser  and  Shiga's  method. — An  emulsion  of  cultures  on  agar  is  made, 
sterilized  at  60°  C.,  macerated  at  37°  C.  for  3  days,  and  then  filtered.  The 
filtrate  without  further  preparation  is  used  as  a  vaccine. 

(7)  Bassenge  and  Mayer's  method. — A  filtrate  of  living  cultures  is  used. 
Make  an  emulsion  in  distilled  water  of  the  growth  of  a  very  virulent  bacillus 
on  agar  and.  after  shaking  continuously  for  3  days,  filter.  A  single  inocula- 
tion is  given  equal  to  the  filtrate  obtained  from  one  tube  of  culture. 

Effects  of  vaccination. 

The  results  obtained  in  man  with  Wright's  and  with  Pfeiffer  and  Kolle's 
vaccines  will  be  chiefly  quoted,  as  these  are  the  best  known  methods  and 
appear  to  give  the  most  satisfactory  results. 

Two  or  three  hours  after  inoculation  tenderness  develops  about  the  site 
of  inoculation,  reaches  its  maximum  in  about  12  hours,  and  vanishes  as  a 
rule  about  40  hours  after  inoculation. 

At  the  same  time  there  is  some  rise  of  temperature  accompanied  by  stiff- 
ness of  the  back  and  limbs,  headache,  loss  of  appetite  and  nausea  lasting 
twenty-four  hours  or  so. 

About  the  end  of  the  first  week  the  serum  has  acquired  bactericidal, 
agglutinating,  bacteriolytic  and  immunizing  properties,  and  the  opsonic 
index  is  raised.  These  newly-acquired  properties  rapidly  increase  and  reach 
their  maximum  on  the  third  day  after  the  second  inoculation. 

The  bactericidal  and  agglutinating  properties  persist  for  a  long  time, 
having  been  demonstrated  18  months  later  by  Bassenge  and  as  long  as  4 
years  afterwards  by  Harrison  and  others.  In  a  person  previously  immunized 
and  whose  serum  no  longer  exhibits  any  appreciable  bactericidal  properties, 
the  inoculation  of  a  very  small  dose  of  vaccine  will  re-create  these  properties 
in  a  very  high  degree  (Wassermann)  :  it  would  therefore  appear  desirable  to 
repeat  the  vaccinating  inoculations  at  intervals  in  order  to  maintain  and 
re-enforce  the  immunity. 

Wright  has  drawn  attention  to  a  fact  which  is  very  important  from  the 
point  of  view  of  prophylaxis.  During  the  first  few  days — less  than  a  week — 
after  inoculation  there  is  a  negative  phase  during  which  the  resistance- 
capacity  of  the  patient  to  the  typhoid  bacillus  is  lowered.  During  this 
period  therefore  vaccinated  persons  should  not  be  exposed  to  infection,  and 
it  follows  that  antityphoid  vaccination  as  now  practised  is  not  permissible  in 
times  of  epidemic  nor  in  endemic  centres  of  the  disease. 

Antityphoid  vaccination  in  the  human  subject  has  been  largely  practised 
in  the  English  and  German  armies.  The  results  are  quite  conclusive  in 
favour  of  vaccination.  Among  the  vaccinated  the  proportion  of  cases  is 
markedly  lower  than  among  the  unvaccinated  ;  moreover,  the  cases  of 
enteric  fever  which  have  been  observed  among  the  vaccinated  have,  as  a 
rule,  been  less  severe  than  among  the  unvaccinated,  and  the  mortality  rate 
is  lower  by  one-half.  The  effects  of  antityphoid  vaccination  last  for  several 
years. 

3.  Besredka's  method. 

The  immunity  conferred  by  the  use  of  antityphoid  serum  being  very 
transitory,  and  the  progress  of  vaccination  with  attenuated  cultures  very 


SERUM  THERAPY  383 

slow  and  irregular,  it  has  been  suggested  by  several  observers  (Leclainche, 
Calmette,  Salimbeni)  that  mixtures  of  specific  serum  and  micro-organisms 
might  prove  more  effective.  The  results  so  far  obtained  have  been  only 
moderately  encouraging,  probably  because  there  has  been  too  much  serum 
in  the  mixtures  used  for  inoculation. 

Bearing  in  mind  the  property  possessed  by  organisms  of  fixing  the  immune 
body  present  in  their  specific  serums,  Besredka  sensitizes  bacilli  with  anti- 
typhoid serum  and  uses  the  sensitized  organisms  for  vaccinating  purposes. 
An  emulsion  of  bacilli  from  a  forty-eight-hour  culture  on  agar  is  made  in 
normal  saline  solution,  mixed  with  antityphoid  serum  and  left  at  37°  C.  for 
24  hours.  The  agglutinated  bacilli  are  then  centrifuged  and  washed  several 
times  with  normal  saline  solution  until  all  traces  of  serum  have  disappeared  ; 
the  emulsion  is  then  heated  in  a  water  bath  at  58°  C.  for  half  an  hour. 

Guinea-pigs  can  be  rendered  highly  immune  in  about  20  hours  by  inocu- 
lating them  sub-cutaneously  with  the  vaccine.  The  immunity  lasts  for 
several  months  (Besredka,  Paladino-Blandini)  and  the  serum  of  the  animals 
is  bactericidal  and  prophylactic. 

In  man,  inoculation  with  Besredka's  vaccine  produces  only  a  very  slight 
tenderness  locally,  and  there  is  ground  for  hoping  that  this  method,  which 
confers  immunity  within  24  hours,  will  in  future  play  an  important  part  in 
the  prophylaxis  of  enteric  fever. 

6.  Serum  therapy. 

Brieger,  and  Wassermann  and  Kitasato,  whose  experiments  have  been  con- 
firmed by  Sanarelli,  Chantemesse  and  Widal  and  others,  have  shown  that 
laboratory  animals  can  be  immunized  against  experimental  infection  by 
inoculating  them  with  the  serum  of  a  vaccinated  animal,  and  that  such  a 
serum  possesses  curative  as  well  as  prophylactic  properties. 

If  a  fatal  dose  of  a  culture  of  the  typhoid  bacillus  be  mixed  with  0'5  c.c.  of  the  serum 
and  inoculated  into  the  peritoneal  cavity  or  beneath  the  skin  of  a  guinea-pig  the 
animal  remains  unaffected. 

Guinea-pigs  can  be  immunized  in  a  few  hours  by  inoculating  them  with  2  c.c.  of 
the  serum  of  a  vaccinated  animal.  Subsequent  inoculation  of  a  dose  of  a  virus 
of  exalted  virulence  sufficient  to  kill  a  control  animal  is  without  effect  on  the  treated 
animal. 

Similarly,  animals  inoculated  with  an  ordinarily  fatal  dose  of  culture  recover  if, 
within  3  hours  of  the  inoculation,  1-2  c.c.  of  antityphoid  serum  be  administered 
to  them. 

Chantemesse  and  Widal  have  shown  that  the  serums  of  patients  who  have 
recovered  from  an  attack  of  enteric  fever  exhibit  both  prophylactic  and 
curative  properties.  These  properties  are  not  very  well  marked,  and  to 
immunize  a  guinea-pig  about  10  c.c.  of  serum  are  necessary.  Attempts  to 
use  the  serum  in  the  treatment  of  human  enteric  fever  have  not  given  conclusive 
results. 

Artificial  animal  serums  have  been  used  by  a  great  many  observers  in  the 
treatment  of  enteric  fever,  but  with  little  result  (Beumer  and  Peipper,  Shaw, 
Tavel,  Aronson,  arid  others). 

Chantemesse  and  Besredka  however  have  prepared  serums  which  un- 
doubtedly possess  therapeutic  powers. 

A.  Chantemesse  s  serum.  1.  Preparation. — Chantemesse  immunizes  horses 
by  repeated  sub-cutaneous  inoculations  of  his  soluble  toxin  (p.  377)  and  intra- 
venous inoculations  of  virulent  typhoid  bacilli.  The  process  of  immuniza- 
tion is  very  lengthy  ;  small  doses  should  be  used  to  begin  with,  and  the  animals 
must  be  carefully  handled. 

2.  Properties. — Chantemesse's    antityphoid    serum    may    be    repeatedly 


384  THE   TYPHOID   BACILLUS 

heated  to  54°-56°  C.  without  losing  any  of  its  properties.  It  shows  marked 
agglutinating  power  (1  in  100,000,  p.  413).  In  vitro  it  has  no  bactericidal 
action,  but  in  vivo  it  stimulates  the  leucocytes  to  take  up  and  dissolve  the 
bacilli .  It  protects  guinea-pigs  and  rabbits  against  the  inoculation  of  lethal 
doses  of  an  exalted  virus.  It  is  markedly  antitoxic  (Balthazard  and  Chante- 
messe),  and  neutralizes  toxin  in  vitro.  If  given  as  a  prophylactic,  2-24  hours 
before  the  inoculation  of  the  toxin,  it  will  protect  rabbits  against  the  effects 
of  four  times  the  lethal  dose  of  toxin.  When  the  serum  is  inoculated  at 
the  same  time  as  the  toxin  but  at  an  independent  site  its  action  is  less  pro- 
nounced, and  animals  which  have  received  more  than  twice  the  lethal  dose 
succumb  (Balthazard).  When  injected  after  the  toxin  the  serum  has  still 
less  prophylactic  and  curative  powers,  and  its  efficacy  varies  inversely  as  the 
time  which  has  elapsed  between  the  inoculation  of  the  toxin  and  the 
inoculation  of  the  serum.  The  prophylactic  properties  of  the  serum 
are  short-lived  and  the  immunity  conferred  lasts  no  longer  than  10  or 
12  days. 

3.  Therapeutic  application. — Chantemesse's  serum  which  in  the  laborator}^ 
shows  only  feeble  curative  power  has  a  marked  influence  on  the  phenomena 
of  opsonization,  and  it  is  probably  to  this  that  its  undoubted  therapeutic 
properties  in  the  treatment  of  enteric  fever  are  due :  according  to  statistics 
published  by  Chantemesse  the  mortality  in  cases  treated  with  the  serum  is 
only  4  per  cent.  It  is  all  important  that  the  serum  should  be  used  in  the 
early  stages  of  the  disease.  Originally  Chantemesse  inoculated  repeated 
doses  of  5-15  c.c.  sub-cutaneously,  but  the  serum  as  now  prepared  is  more 
active  and  a  single  inoculation  of  a  few  drops  is  sufficient.  • 

B.  Besredka's  serum. — An  anti-endotoxic  serum  has  been  prepared  by 
Besredka,  by  inoculating  killed  cultures  followed  by  living  cultures  of  the 
bacillus  into  the  veins  of  animals. 

The  serum  neutralizes  ten  to  twenty  fatal  doses  of  Besredka's  endotoxin 
and  acts  as  a  prophylactic  to  the  inoculation  of  the  endotoxin. 

Montefusco,  who  has  used  Besredka's  serum  in  the  treatment  of  enteric 
fever  at  Naples,  has  obtained  very  satisfactory  results,  and  thinks  it  will  be 
of  great  value  in  the  treatment  of  the  disease. 

7.  Agglutination.    Serum-diagnosis  of  enteric  fever. 

Durham  and  Gruber  were  the  first  to  show  that  [an  antiserum]  agglu- 
tinates [its  homologous  organism].  This  agglutinating  property,  which  is  a 
reaction  of  infection  (p.  225),  is  also  manifested  in  the  blood  of  persons 
suffering  from  or  who  have  recovered  from  an  attack  of  enteric  fever. 
Agglutination  can  also  be  obtained  with  the  blister  fluid,  milk,  naturally 
shed  tears,  and  occasionally  even  with  pus,  urine,  bile,  etc.  from  these 
persons. 

[A.  S.  Griinbaum  first  and]  Widal  [afterwards]  utilized  the  agglutinat- 
ing properties  of  the  blood  of  enteric  fever  patients  as  a  rapid  and  conclusive 
method  of  diagnosis — -the  serum  diagnosis  of  enteric  fever. 

The  power  of  agglutinating  the  typhoid  bacillus  is  developed  in  the  blood 
of  the  patient  as  a  rule  in  the  early  days  of  the  illness,  and  while  it  may  not 
infrequently  be  delayed,  it  is  only  very  exceptionally  that  it  is  absent  through- 
out the  whole  course  of  the  disease  ;  (Widal  and  Sicard  failed  to  get  agglutina- 
tion once  only  in  163  cases  :  Besson  twice  in  98  cases).  The  power  of 
agglutination  may  disappear  during  the  early  weeks  of  convalescence,  and  is 
generally  absent  6-8  months  after  recovery  ;  but  occasionally  it  has  been 
present  as  long  as  3  and  even  7  years  after  the  attack. 

A  positive  result  obtained  under  the  conditions  to  be  immediately  described 


GRUNBAUM-WIDAL  REACTION  385 

may  be  taken  as  a  certain  indication  of  enteric  fever.1  On  the  other  hand 
a  negative  result  establishes  merely  a  probability  that  the  disease  is  not 
enteric  fever.  A  negative  result  in  the  early  days  of  a  suspected  attack  of 
the  disease  is  of  less  value  than  one  obtained  later,  for  if  the  disease  be  enteric 
fever  failure  to  react  is  then  improbable.  But  in  any  case  if  the  result  be 
negative  opportunity  should  always  be  taken  to  test  the  blood  again  later. 

The  reaction  may  be  performed  in  several  ways  which  are  described  as 
slow  or  rapid  according  to  the  time  required  :  but  whatever  the  method 
adopted  the  following  rules  must  be  observed. 

General  rules. — 1.  For  the  slow  methods  the  blood  must  be  taken  under 
aseptic  precautions  from  a  vein  at  the  bend  of  the  elbow  (p.  193),  and  may  be 
conveniently  collected  in  small  sterile  glass  tubes.  For  the  rapid  methods 
sufficient  blood  can  be  collected  in  capillary  tubes  by  pricking  the  finger 
(p.  192). 

2.  When  the  blood  has  to  be  sent  some  distance  to  a  laboratory  the  tube 
in  which  it  has  been  collected  should  be  plugged  with  a  plug  of  wool  passed 
through  the  flame.     Serum  kept  in  the  liquid  condition  retains  its  power 
of  agglutination  for  a  very  long  time.     Since,  however,  drying  has  no  effect 
on  the  agglutinating  power  of  the  blood,  a  few  drops  of  the  latter  may  be 
collected  on  a  piece  of  paper  or  on  a  glass  slide  and  allowed  to  dry  before 
being  sent  to  the  laboratory,  and  in  some  cases  this  may  be  the  more  con- 
venient course  to  adopt.     For  purposes  of  the  agglutination  reaction  the 
dried  blood  is  dissolved  in  a  drop  or  two  of  sterile  water. 

In  the  author's  experience  good  results  have  always  been  obtained  when  the 
blood  was  dried  on  glass  but  when  dried  on  paper  it  seemed  to  lose  some  of  its 
agglutinating  power.  Dried  blood  is  only  available  for  use  by  the  rapid  method. 

3.  The  culture  should  always  be  examined  microscopically  to  test  its  purity 
immediately  before  being  used  for  the  reaction.     The  mistakes  which  might 
arise  from  the  use  of  an  impure  culture  can  be  readily  appreciated. 

4.  The  serum  must  be  added  to  the  culture  and  not  vice  versa  (p.  226). 

A.  Slow  method. — The  blood  (collected  from  a  vein  at  the  bend  of  the 
elbow  to  ensure  that  a  sufficient  quantity  is  obtained)  must  be  absolutely 
pure  and  uncontaminated.  and  after  collection  should  be  set  aside  in  a  sterile 
tube  until  the  clot  has  separated  ;  the  serum  is  then  drawn  up  into  a  Pasteur 
pipette. 

1.  To  a  tube  containing  6-10  c.c.  of  sterile  broth,  add  ten  drops  of  the 
serum  and  sow  with  a  trace  of  a  culture  of  the  typhoid  bacillus.  A  control 
consisting  of  a  tube  of  broth  containing  no  serum  but  sown  with  a  trace  of 
a  typhoid  culture  must,  of  course,  be  put  up.  Incubate  the  tubes  at  37°  C. 
Growth  in  the  tube  to  which  the  serum  has  been  added  will  be  somewhat 
delayed,  but  small  clumps  appear  after  8  hours  or  so,  and  after  about  18 
hours'  incubation  the  appearance  is  characteristic  :  the  bacilli  are  collected 
together  at  the  bottom  of  the  tube  in  little  whitish  flocculi,  which  cannot  be 

1  In  suspected  cases,  the  possibility  of  the  patient  having  had  previously  an  attack  of 
enteric  fever  should  always  be  borne  in  mind,  because  if  so  the  blood  might  still  retain 
some  agglutinating  power.  A  few  rare  cases  are  on  record  in  which  it  was  found  that 
the  blood  of  persons  suffering  from  diseases  other  than  enteric  fever  has  agglutinated 
the  typhoid  bacillus  in  dilutions  of  1  in  100  and  1  in  250.  Thus  in  an  undoubted  case  of 
pneumonia  in  a  young  man  in  which  there  was  no  reason  to  suspect  a  previous  attack  of 
enteric  fever  Besson  found  that  the  blood  of  the  patient  agglutinated  the  typhoid  bacillus. 
The  serum  reaction  in  the  absence  of  enteric  infection  has  also  been  observed  in  a  case  of 
tuberculous  meningitis  (E.  Mackey)  and  in  a  case  of  abscess  of  the  liver  (Megele).  In 
pathological  conditions  in  which  the  bile  enters  the  blood  stream,  as  for  instance  in 
jaundice  or  occlusion  of  the  bile-duct,  the  blood  may  agglutinate  the  typhoid  bacillus 
(Griinbaum,  Zupnik,  Kohler  and  others). 

2B 


386  THE   TYPHOID   BACILLUS 

broken  up  by  shaking  the  tube,  while  the  broth  remains  perfectly  clear. 
In  the  control  tube,  on  the  other  hand,  the  broth  is  cloudy  and  shows  the 
scintillating  ripples  characteristic  of  a  growth  of  the  typhoid  bacillus. 

The  reaction  is  not  always  so  distinct  as  this.  Sometimes  the  broth  instead  of 
remaining  clear  shows  an  irregular  turbidity,  from  which  however  the  characteristic 
watered-silk  appearance  is  absent ;  this  turbidity  is  due  to  the  precipitation  of  a 
very  fine  powder  each  grain  of  which  when  examined  under  the  microscope  is  seen 
to  be  an  agglomeration  of  bacilli.  At  other  times  the  reaction  may  be  quite  charac- 
teristic at  first  but  after  incubating  for  18  or  24  hours  the  broth  is  turbid  above 
the  precipitate.  Naked  eye  appearances  ought  always  to  be  supplemented  by  a 
microscopical  examination  by  means  of  which  the  small  masses  of  bacilli  may  be 
recognized  if  present  and  their  structure  defined. 

2.  Add  the  serum  to  a  twenty-four-hour  broth  culture  of  the  typhoid 
bacillus  and  incubate  at  37°  C.  If  the  agglutinating  power  of  the  serum  is 
well  marked  characteristic  changes  will  take  place  within  a  few  hours  ;  the 
culture  is  at  first  granular,  but  becomes  gradually  clear  as  the  bacilli  fall  to 
the  bottom.  When  the  serum  is  less  powerfully  agglutinating,  clumps  are 
formed  but  the  broth  never  becomes  quite  clear.  Naked  eye  appear- 
ances must,  as  in  the  previous  case,  always  be  controlled  by  microscopical 
examination. 

B.  Rapid  method.  Recommended. — This  method  is  quicker  and  more 
sensitive  than  the  foregoing  and  has  the  additional  advantage  of  requiring 
only  a  few  drops  of  blood,  an  amount  which  can  be  easily  obtained  by  prick- 
ing the  finger.  It  is  therefore  the  method  to  be  used  in  the  majority  of  cases. 

A  broth  or  peptone  culture  of  the  typhoid  bacillus  is  required  for  the  reac- 
tion, and  the  greatest  care  should  be  exercised  in  the  choice  of  a  culture. 
In  the  first  place  it  is  of  course  essential  that  it  be  pure  :  secondly  the  growth 
must  not  be  more  than  24  hours  old,  because  in  old  cultures  clumps  often 
form  spontaneously  and  these  will  falsify  the  results.  Spontaneously  formed 
clumps  may  indeed  be  present  even  in  twenty-four-hour  cultures,  so  it  is 
always  necessary  to  determine  by  microscopical  examination  immediately 
before  use  that  the  chosen  culture  is  satisfactory  in  this  respect.  A  quantity 
of  culture  sufficient  for  the  investigation  should  be  drawn  up  into  a  Pasteur 
pipette  and  a  little  placed  on  a  slide  and  examined  under  the  microscope, 
the  remainder,  if  the  sample  is  satisfactory,  being  used  for  the  serum  reaction. 
To  obviate  the  spontaneous  formation  of  clumps  in  cultures  it  is  better  to 
grow  the  organism  in  a  1  or  2  per  cent,  solution  of  peptone  containing  no 
meat  rather  than  in  broth. 

The  method  is  as  follows. 

1.  Reaction  with  the  serum.— [(a)  Technique  recommended,—!.  Take  a 
sterile  Pasteur  pipette,  plugged  with  wool  and  fitted  with  an  india-rubber  teat 
as  shown  in  fig.  162,  p.  241.  Make  a  mark  on  the  stem  of  the  pipette. 

[2.  Take  up  9  volumes  of  the  peptone  water  culture  run  them  up  into  the 
bulb  and  then  take  up  1  volume  of  the  serum.  Mix  the  culture  and  serum 
thoroughly  by  repeatedly  expelling  on  to  a  slide  and  aspirating.  (Dilution  1.) 

[3.  Take  up  4  volumes  of  dilution  1  and  1  volume  of  serum.  Mix. 
(Dilution  2.— 1  in  50.) 

[4.  Take  up  equal  volumes  of  dilution  2  and  culture.  Mix.  (Dilution  3. 
—1  in  100.) 

[And  so  on,  preparing  the  dilutions  required. 

[Place  a  drop  of  each  dilution  on  a  clean  cover -glass  and  invert  the  latter 
over  the  cavity  in  a  hollow-ground  slide.  Lute  the  edge  with  vaseline.  Place 
the  preparations  in  the  incubator  at  37°  C.  Examine  with  a  high  power  dry 
lens  at  the  end  of  half  an  hour  and  again  at  the  end  of  an  hour.  If  the  serum 


GRUNBAUM-WIDAL  REACTION  387 

be  a  typhoid-agglutinating  serum  the  bacilli  will  be  found  agglutinated  together 
in  more  or  less  large  masses.] 

(/3)  Another  method. — Into  a  small  conical  glass  vessel  introduce  10-100 
drops  of  the  culture  and  1  drop  of  the  serum.  Place  a  drop  of  the  mixture 
on  a  slide  and  cover  with  a  cover-glass.  Examine  with  a  high  power  dry 
lens.  If  the  serum  has  the  power  of  agglutinating  the  typhoid  bacillus, 
masses  of  agglutinated  bacilli  will  be  seen  and  among  them  a  greater  or 
smaller  number  of  non-agglutinated  bacilli.  The  reaction  is  still  more 
distinct  if  the  preparation  be  examined  after  15  or  20  minutes,  for  compact 
islets  of  agglutinated  bacilli  will  then  be  visible  under  the  microscope.  When 
the  agglutinating  property  of  the  serum  is  small  the  reaction  may  only  appear 
after  the  lapse  of  40  minutes  or  an  hour.  The  appearance  is  quite  charac- 
teristic and  renders  mistakes-  impossible.  Agglutination  is  assisted  by  a 
slight  drying  at  the  edge  of  the  drop  between  the  slide  and  the  cover- 
glass. 

Whole  blood. — The  whole  blood  may  be  used  for  the  reaction,  but  before  examining 
the  preparation  under  the  microscope  time  must  be  given  to  allow  most  of  the  red 
cells  to  settle,  since  the  presence  of  a  large 
number  of  cells  detracts  from  the  sharp- 
ness of  the  reaction.     The  method  is  there- 
fore no  quicker  than  the  serum  method. 

Method  of  staining. — The  preparation 
may  be  stained — so  as  to  render  the  masses 
more  distinct — and  preserved  for  future 

use:  for  this  purpose  the  folio  wing  technique,  _ 

described  by  Guillemin,  gives  good  results. 

Mix  1  drop  of  the  whole  blood  with  9 
drops  of  sterile  broth,  and  add  1  drop  of 
this  to  2-5  drops  of  a  culture  of  the 
typhoid  bacillus.  Spread  a  large  drop  of 
the  mixture  on  a  slide :  place  in  a  moist 
chamber  for  an  hour  or  two  :  dry  slowly, 
fix  in  alcohol-ether,  treat  with  10  per  cent, 
acetic  acid  to  dissolve  the  red  cells,  wash, 
stain  with  dilute  carbol-fuchsin,  wash  and 

C^r'  FIG.  220.— Agglutination  of  the  typhoid  bacillus 

2.  Reaction  with  dried  blood.-The  ^VS  serum<   Jenner>S  ^   (°C-  2> 
blood  collected   and   dried  as  already 

described  is  dissolved  immediately  before  use  in  a  drop  or  two  of  water.  The 
solution  is  added  to  10-50  drops  of  a  broth  culture  of  the  typhoid  bacillus 
contained  in  a  conical  glass  vessel.  It  is  left  for  a  moment  to  allow  the  red 
cells  to  settle  and  then  examined  as  before. 

3.  Reaction  with  dead  bacilli. — The  phenomenon  of  agglutination  is  not 
dependent  upon  any  vital  reaction  of  the  bacilli,  since  it  can  be  demon- 
strated with  dead  organisms.     This  fact  may  in  certain  circumstances  be  of 
practical  value,  because  a  recent  culture  of  the  typhoid  bacillus  is  not  always 
immediately  available  with  which  to  perform  the  reaction,  and  to  obtain 
one  may  involve  a  delay  of  12  hours  or  so.     In  such  a  case  a  dead  culture  may 
be  used,  since  experience  has  shown  that  such  a  culture  retains  its  sensitive- 
ness  towards   an   agglutinating   serum   for   several   weeks.     The   following 
technique  may  be  adopted  in  the  preparation  of  a  dead  culture  for  this  purpose 
(Widal  and  Sicard). 

A  sixteen-  or  twenty-four-hour  culture  of  the  typhoid  bacillus  is  examined  micro- 
scopically to  test  its  purity.  Formalin  in  the  proportion  of  2  drops  to  15  c.c.  of  culture 
is  added  to  kill  the  bacilli,  which  become  as  it  were  embalmed.  Care  must  be  taken 
to  cover  the  cotton- wool  plug  of  the  vessel  containing  the  culture  with  an  india- 


388  THE   TYPHOID   BACILLUS 

rubber  cap.  Cultures  killed  in  this  way  may  be  stored  exactly  as  chemical  reagents 
are  stored  in  the  laboratory.  Immediately  before  use  the  tube  is  lightly  shaken 
so  that  the  bacilli  shall  be  uniformly  distributed  through  the  medium.  To  a  few 
drops  of  this  dead  culture  a  few  drops  of  serum  are  added  in  the  same  manner  as 
has  been  described  above. 

There  are  now  a  number  of  preparations  of  dead  bacilli  on  the  market 
such  as  Ficker's  "  Typhus  diagnosticum,"  Stassano's  emulsion,  etc.  These 
preparations  allow  the  practitioner  to  perform  a  serum  diagnosis  rapidly 
and  easily.  The  "  Typhus  diagnosticum  "  of  Ticker  in  particular  has  given 
good  results  in  the  hands  of  many  bacteriologists — though  its  reliability  is 
questioned  by  de  Rossi. 

De  Rossi  advises  the  use  of  broth  cultures  which  have  been  heated  to 
58°-60°  C.  for  an  hour.  The  resulting  emulsion  agglutinates  more  readily 
than  unheated  cultures  and  preserves  its  property  for  at  least  3  months. 

Tribondeau  uses  broth  cultures  killed  by  the  addition  of  formalin  (1-150) 
and  stored  in  sealed  ampoules.  Under  these  conditions  the  bacilli  retain 
their  capacity  for  agglutination  for  4  years  and  more. 

Measurement  of  the  agglutinating  titre.— The  serums  of  different  patients 
vary  in  their  agglutinating  powers  ;  sometimes  this  power  is  very  feeble 
while  in  other  cases  it  is  so  well  marked  that  clumps  are  formed  when  the 
serum  is  diluted  as  much  as  1-5000  and  1-15,000  (Jurgens). 

In  investigating  the  agglutinating  property  of  a  given  serum  the  examina- 
tion should  be  begun  with  a  dilution  of  1-10.  Agglutination  in  a  lower 
dilution  than  this  is  in  no  way  characteristic,  and  indeed,  since  normal  human 
serum  occasionally  agglutinates  when  diluted  1  in  10  or  even  1  in  20  (vide 
colon  bacillus),  a  reliable  diagnosis  can  according  to  Remy  only  be  given 
when  agglutination  is  found  with  a  dilution  of  1  in  50.  [Moreover  with  some 
serums  there  appears  to  be  an  agglutination-inhibiting  action  when  examined 
in  low  dilutions.  ]  The  degree  to  which  the  agglutinating  power  is  developed 
should  therefore  be  measured  more  exactly  by  investigating  dilutions  of 
1  in  20,  1  in  30  [and  so  on  to  at  least  a  dilution  of  1-100]. 

In  practice  when  but  a  small  quantity  of  blood  is  available  two  tests  suffice, 
one  made  with  a  dilution  of  1  in  10  the  other  with  a  dilution  of  1  in  50.  [By 
the  method  described  above  (B.  1.  a)  if  a  dilution  of  -1  in  10  can  be  obtained 
a  dilution  of  1  in  100  and  1  in  500  can  also  be  made.  In  our  experience  a 
dilution  of  1  in  100  is  the  smallest  dilution  upon  which  a  reliable  opinion  can 
be  based  in  suspected  cases  of  enteric  fever.] 

Widal  and  Sicard  draw  the  following  distinctions. 

Agglutinating  power  very  feeble  if  exhibited  only  in  dilutions  below  1  in  100. 
Agglutinating  power  feeble  if  exhibited  only  in  dilutions  between  1  in  100  and  1 

in  200. 
Agglutinating  power  average  if  exhibited  in  dilutions  1  in  200  and  1  in  500 

„      marked  „  „  1  in  500  and  1  in  2000. 

„  ,.      very  marked  „  „  above  1  in  2000. 

Note. — In  these  measurements  it  is  important  that  the  drops  of  culture  and  of 
the  serum  be  equal  in  size.  A  sufficient  degree  of  accuracy  is  attained  by  the  follow- 
ing method :  take  a  piece  of  glass  tubing  about  20  cm.  long  and  plug  it  at  both 
ends  with  wool.  Draw  it  out  in  the  flame  as  though  making  a  Pasteur  pipette. 
Sterilize  the  tube  without  cutting  it  into  two  and  then,  when  about  to  use  it,  file  it 
in  the  middle  of  the  capillary  portion.  In  this  way,  two  pipettes  are  obtained, 
which  for  all  practical  purposes  will  give  drops  of  equal  size  :  one  will  serve  for 
the  culture,  the  other  for  the  serum. 

From  the  point  of  view  of  prognosis,  it  appears  that  the  degree  to  which 
the  agglutinating  power  is  developed  [has  no  consistent  relation  to,  and 
therefore]  furnishes  no  reliable  information  as  to  the  severity  of  the 
disease. 


ABSORPTION   OF  AGGLUTININS  389 

[Co-agglutinins  in  the  serums  of  enteric  patients.] 

[In  addition  to  the  specific,  homologous  or  primary  agglutinins  for  the 
typhoid  bacillus,  the  serum  of  enteric  fever  patients  often  contains  group  or 
heterologous  or  secondary  agglutinins  for  bacilli  of  the  paratyphoid  and 
salmonella  groups. 

[In  a  consecutive  series  of  86  serums  Boycott  found  that  59  per  cent,  con- 
tained secondary  agglutinins  and  of  this  series  55  per  cent,  reacted  with 
B.  Gaertner  and  Paratyphosus  A  (Brion  and  Kayser),  41  per  cent,  with  Para- 
typhosus  B  (Schottmiiller),  33  per  cent,  with  Aertrycke  and  12  per  cent,  with 
Paratyphosus  B  (Schottmiiller).  Generally  speaking  the  more  typhoid 
agglutinin  there  is  present,  the  more  secondary  agglutination  is  likely  to 
be  found.] 

The  application  of  the  serum  test  to  the  identification  of  the 
typhoid  bacillus. 

The  application  of  the  agglutination  reaction  to  the  identification  of  the 
typhoid  bacillus  may  be  of  considerable  service  but  is  not  a  test  sufficiently 
delicate  and  specific  to  determine  the  identity  of  the  bacillus  with  certainty. 

For  the  purpose  of  testing  whether  a  given  organism  be  the  typhoid  bacillus 
or  no  it  is  better  to  use  the  serum  of  a  person  suffering  from  the  disease  which 
agglutinates  quite  distinctly  in  a  dilution  of  1  in  100  than  an  artificially 
prepared  anti-typhoid  serum.1  Typical  typhoid  bacilli  are  agglutinated  by 
this  serum  in  dilutions  varying  from  1  in  50  to  1  in  100.  Strains  of  the 
colon  bacillus  on  the  other  hand  are  never  agglutinated,  or  at  most  only  in 
dilutions  of  1  in  5  to  1  in  10.  A  postulate  such  as  the  following  would  render 
the  diagnosis  very  simple  :  any  bacillus  agglutinated  in  a  dilution  of  1  in  50 
may  be  legitimately  described  as  a  typhoid  bacillus. 

Unfortunately,  it  is  now  established  that  there  are  some  undoubted  typhoid 
bacilli  which  are  not  agglutinated  by  the  serum  of  a  person  suffering  from 
enteric  fever.  Remy  has  shown  that  a  typhoid  bacillus  which  agglutinated 
well  at  first  readily  lost  this  property  when  grown  symbiotically  with  the  colon 
bacillus  for  a  few  weeks.  Occasionally,  strains  of  the  typhoid  bacillus  isolated 
from  the  living  body  or  from  water  can  be  agglutinated  only  with  difficulty, 
and  it  is  not  until  they  have  been  sub -cultivated  a  certain  number  of  times 
on  artificial  culture  media  that  agglutination  capacity  is  acquired  (Courmont, 
Chantemesse,  Remy,  Sacquepee  and  others).  When  a  suspected  typhoid 
bacillus  has  failed  to  give  the  serum  reaction  the  following  experiment  may 
be  carried  out  with  the  object  of  identifying  the  organism.  Inoculate  a 
guinea-pig  every  other  day  for  a  fortnight  with  2  c.c.  of  a  forty-eight-hour 
broth  culture  of  the  bacillus  under  investigation.  If  the  blood  of  the  guinea- 
pig  now  agglutinates  an  undoubted  typhoid  bacillus  in  a  minimum  dilution 
of  1  in  40  the  organism  which  served  for  the  inoculation  of  the  animal  may 
be  regarded  as  a  true  typhoid  bacillus.  Some  strains  of  undoubted  typhoid 
bacilli  however  escape  even  this  method  of  recognition  (Remy). 

8.  Absorption  of  agglutinins. 

Castellani's  absorption  or  saturation  method  can  also  be  applied  to  the 
differentiation  of  the  typhoid  bacillus  from  closely  allied  organisms  and  by 

1  In  the  case  of  animals  highly  immunized  against  the  typhoid  bacillus,  Rodet  has 
shown  that  the  blood  not  only  agglutinates  the  typhoid  bacillus  in  very  high  dilutions, 
but  that  it  also  has  marked  agglutinating  properties  for  some  strains  of  the  colon  bacillus. 
Pfaundler,  Bruns,  Kayser,  have  shown  that  very  highly  immunized  serums  agglutinate 
not  only  the  organism  against  which  the  animals  were  immunized  but  also  closely  related 
species. 


390  THE  TYPHOID   BACILLUS 

its  means  it  is  possible  to  determine  whether  agglutinins  which  have  been 
detected  in  a  suspected  typhoid  serum  are  specific  agglutinins  or  co-agglu- 
tinins.  (For  technique  see  p.  436.) 

9.   Complement  fixation. 

The  method  of  complement  fixation  (Bordet-G-engou  reaction)  is  applicable 
to  the  diagnosis  of  enteric  fever  and  to  the  identification  of  the  typhoid 
bacillus.  The  method  is  described  at  p.  233.  The  results  are  more  exact 
and  more  reliable  than  agglutination  (Widal  and  Le  Sourd)  and  the  reaction 
gives  positive  results  with  the  serum  of  "  carriers  "  even  when  no  bacilli 
can  be  detected  (Scheme). 

[H.  K.  Dean  finds  that  the  complement-fixation  method  affords  an  extremely 
delicate  and  specific  means  of  differentiating  between  various  members  of  the 
typhoid  and  paratyphoid  group.  (For  Dean's  technique  see  p.  428).] 

SECTION  IV.— DETECTION,  ISOLATION  AND  IDENTIFICATION   OF 
THE  TYPHOID   BACILLUS. 

The  detection  of  the  typhoid  bacillus  may  be  rendered  difficult  by  the 
presence  of  other  organisms  in  the  fluid  or  tissue  under  examination.  Thus, 
in  patients  suffering  from  enteric  fever  or  in  patients  or  animals  who  have 
died  from  the  infection  the  bacillus  occurs  in  pure  culture  and  can  be  readily 
isolated,  but  when  it  is  necessary  to  isolate  it  from  water,  dust,  stools,  etc.  the 
presence  of  the  colon  bacillus  often  renders  the  investigation  by  no  means  easy. 

The  methods  of  isolating  the  typhoid  bacillus  from  water  and  other  sources 
is  dealt  with  in  a  separate  chapter  (Chap.  XXIII.,  p.  401)  and  here  the  more 
simple  investigations  only  will  be  considered  in  which  the  organism  is  assumed 
to  be  in  pure  culture  in  a  fluid  or  tissue  of  the  body. 

1.  Microscopical  examination. 

For  purposes  of  microscopical  examination  films  and  sections  of  the  spleen 
and  other  organs  as  well  as — in  the  case  of  experimentally  infected  animals— 


FIG.  221.— Typhoid  bacillus.     Section  of  a  spleen.    Carbol-thionin. 
(Oc.  2,  obj.  TUh,  Zeiss.) 

films  from  the  pus  of  typhoid  abscesses  and  the  peritoneal  exudate,  should 
be  made. 


DETECTION   OF  THE   BACILLUS  391 

In  no  case  can  an  absolute  diagnosis  be  made  on  microscopical  evidence 
alone. 

(a)  Films. — Films  should  be  stained  first  with  methylene  blue  or  carbol- 
thionin  and  then  by  Gram's  method. 

Films  from  the  spleen  often  contain  only  a  few  organisms,  but  if  the  tissue  be  first 
incubated  films  made  from  it  will  be  found  to  be  very  rich  in  bacilli.  Wash  the 
surface  of  the  organ  in  a  1  in  1,000  solution  of  perchloride  of  mercury,  wrap  it  up  in 
a  cloth  wrung  out  of  the  same  solution  and  incubate  at  37°  C.  for  24  hours  (Cornil) ; 
or  if  preferred  a  little  of  the  pulp  may  be  drawn  up  into  a  number  of  Pasteur  pipettes 
and  incubated  (Gasser). 

Gasser  prefers  to  stain  the  films  obtained  in  this  way  by  Gram's  method,  using 
dilute  carbol-fuchsin  as  a  counterstain :  the  typhoid  bacilli  and  the  groundwork 
are  then  stained  red  while  if  any  gram-positive  organisms  are  present  they  are,  of 
course,  stained  violet. 

(b)  Sections.— Fix  the  tissues  to  be  cut  in  alcohol  or  acid  perchloride  solu- 
tion (p.  189)  and  embed  in  paraffin.     Stain  the  section  by  any  of  the  methods 
applicable   to   the   staining   of  gram-negative   organisms — preferably   with 
thionin  or  by  Nicolle's  tannin  method  (p.  217). 

2.  Cultures. 

Culture  media — broth,  agar  (and,  for  isolating  the  bacillus  when  con- 
taminations are  present,  gelatin  plates) — should  be  sown  with  scrapings 
from  the  spleen,  with  fluid  exudates,  products  from  puncture  of  the  tonsil, 
urine  collected  under  aseptic  precautions,  etc. 

Attention  has  already  been  drawn  (p.  198)  to  the  dangers  attending  puncture  of  the 
spleen  in  the  living  subject.  This  practice  should  never  be  resorted  to  as  a  matter 
of  routine  and  since  the  serum  reaction  is  now  available  for  the  purposes  of  diagnosis 
(see  p.  384)  and  the  bacilli  can  be  isolated  from  the  blood  there  is  no  justification  for 
running  the  risk  attending  the  operation. 

Examination  of  the  blood,  (a)  Courmont's  method. — Collect  the  blood 
aseptically  by  puncture  of  a  vein  at  the  bend  of  the  elbow  (p.  193)  and  sow 
2-4  c.c.  immediately  in  a  large  volume  (200-300  c.c.)  of  ordinary  broth. 
Incubate  at  37°  C.  If  no  turbidity  appears  after  24  hours  shake  the  flask 
to  promote  the  growth  of  the  organism  and  then  incubate  again,  and  examine 
the  culture  daily.  In  some  cases  when  the  typhoid  blood  has  powerful 
agglutinating  properties  growth  may  be  delayed,  being  invisible  until  the 
third  or  fourth  day,  and  may  then  occur  solely  as  clumps  in  the  deposit  which 
forms  at  the  bottom  of  the  liquid. 

(/?)  Busquet's  method. — To  minimize  the  inconvenience  caused  by  the  agglutinating 
and  bactericidal  properties  of  the  blood,  Busquet  sows  a  number  of  flasks  each  con- 
taining 250  c.c.  of  peptone  broth  with  a  few  drops  only  of  blood. 

Sacquepee  and  Perquis  adopt  the  additional  precaution  of  defibrinating  the 
blood  as  it  leaves  the  vein. 

(y)  Laff  orgue  s  method. — Lafforgue  eliminates  the  serum,  which  contains  the 
bactericidal  substances.  The  blood  is  rendered  non-coagulable  by  the  addition  of 
sodium  citrate  (2  drops  of  a  1  in  5  solution  of  sodium  citrate  to  2  c.c.  of  blood)  and 
then  centrifuged.  The  deposit  only  is  sown  in  broth  in  the  proportion  of  20  c.c.  of 
broth  to  the  deposit  from  2  c.c.  of  blood.  Under  these  conditions  the  typhoid 
bacillus  grows  rapidly. 

(S)  Conradi's  method. — Conradi  has  shown  that  bile  is  an  excellent  medium, 
since  it  renders  the  blood  non-coagulable  and  inhibits  its  bactericidal  action. 
Cultures  in  bile-containing  media  are  recommended  for  the  detection  of  the 
typhoid  bacillus  in  blood. 

The  principle  may  be  applied  in  different  ways.  The  simplest  methods 
are  those  of  Zeidler  and  Kayser. 

1.  Methods  of  Zeidler  and  Kayser. — Zeidler  adds  1  c.c.  of  blood  (30  drops 


392  THE   TYPHOID   BACILLUS 

to  5  c.c.  of  ox  bile  previously  sterilized  in  the  autoclave.  The  mixture  is 
incubated  at  37°  C.  for  12-24  hours  and  then  plated  out  on  malachite  green 
agar  (p.  409)  or  better,  litmus-lactose-agar. 

Kayser  adopts  a  similar  technique  and  sows  2*5  c.c.  of  fresh  blood  in  5  c.c. 
of  sterile  ox  bile  and  then  plates  on  Conradi-Drigalski's  medium  (p.  407). 

2.  Conradi's  technique. — Conradi  uses  ox  bile  containing  10  per  cent,  of  peptone 
and  10  per  cent,  of  glycerine.     The  mixture  is  sterilized  at  100°  C.  for  2  hours.     A 
small  clot  of  the  suspected  blood  is  added  to  5  c.c.  of  the  sterilized  medium  and  incu- 
bated at  37°  C.  for  about  15  hours.     Plates  of  litmus-lactose-agar  are  then  sown  with 
the  cultures. 

3.  Roosen-Runge's  technique. — Glycocholate  of  sodium  is  used  instead  of  bile. 
The  medium  is  an  ordinary  agar  medium  to  which  10  grams  per  litre  of  glycocholate 
of  sodium  have  been  added. 

4.  Dlinschmann's  technique. — Dlmschmann  is  of  opinion  that  the  valuable  con- 
stituent in  bile  is  taurocholate  and  not  glycocholate  of  sodium.     He  recommends 
the  following  medium  : 

Gelatin,  -  5  grams. 

Agar,       -  30 

Lactose,  -  40        „ 

Peptone,  10         „ 

Taurocholate  of  sodium,  -                                                                      20        ,, 

Water,     -         -  -       1000  c.c. 

When  only  a  small  quantity  of  blood  is  available  it  is  used  for  sowing  surface 
plates  on  bile-salt-agar. 

Detection  in  sputum. — To  detect  the  typhoid  bacillus  in  sputum  employ 
one  of  the  methods  described  in  Chau.  XXIII. 


CHAPTER  XXII 
BACILLUS  COLL 

Introduction. 

Section  I. — Experimental  inoculation,  p.  394. 

Section  II. — Morphology  and  cultural  characteristics,  p.  395. 

Section  III. — Biological  properties,  p.  396. 

1.  Bio-chemical  reactions,  p.  396.     2    Variability  of  flagella,  p.  398.     3.  Vitality 

and  virulence,  p.  398.     4.  Toxin,  p.  398.     5.  Vaccination  and  serum  therapy,  p.  399. 

6.  Agglutination,  p.  399. 
Section  IV. — Detection,  isolation  and  identification  of  the  colon  bacillus,  p.  400. 

The  bacillus  of  green  diarrhoea,  p.  400. 

IN  man  and  the  lower  animals  the  colon  bacillus,  which  was.  originally 
described  by  Escherich,  is  a  normal  inhabitant  of  the  alimentary  canal  where 
it  makes  its  appearance  a  few  hours  after  birth. 

In  the  intestines  of  healthy  human  subjects  the  colon  bacillus  is  associated 
with  numerous  other  micro-organisms  (at  least  fifty  different  species  are 
present,  many  of  them  being  anaerobes)  ;  it  is  also  frequently  found  in  the 
mouth — twenty-five  out  of  sixty-five  cases  (Grimbert  and  Choquet). 

Though  often  only  slightly  virulent  when  isolated  from  the  healthy  intestine, 
the  colon  bacillus  in  certain  circumstances  and  in  a  diseased  environment 
may  acquire  a  high  degree  of  virulence,  as  for  instance  in  all  febrile  con- 
ditions, in  enteric  fever,  and  in  the  majority  of  diseases  of  the  intestine  ;  and 
may  then  act  as  the  causal  agent  of  a  number  of  diseases  affecting  man. 

It  is,  for  instance,  the  cause  of  secondary  infections  in  enteric  fever,  dysentery 
and  cholera. 

In  some  cases  of  septicaemia  the  colon  bacillus  is  the  organism  present  in  the 
blood,  and  while  as  a  rule  bacillaemic  conditions  due  to  it  are  not  severe,  they  may  on 
occasions  present  all  the  clinical  features  of  enteric  fever.  The  bacillus  is  also  the 
cause  of  some  attacks  of  enteritis,  of  some  cases  of  choleraic  and  infantile 
diarrhoaa,  etc.  Peritonitis  may  sometimes  be  due  to  the  colon  bacillus  (as,  for 
instance,  peritonitis  resulting  from  perforation  of  the  gut  or  following  strangulated 
hernia,  and  peritonitis  unaccompanied  by  perforation).  Invading  the  biliary 
passages  this  organism  determines  suppurative  cholangitis  and  possibly  infective 
jaundice,  and  is  responsible  for  some  cases  of  sore  throat,  broncho-pneumonia, 
endocarditis,  pericarditis,  and  meningitis.  It  is  the  causal  agent  in  a  number  of 
infections  of  the  urinary  passages  and  must  be  identified  with  the  urinary  bacillus 
of  Clado.  In  women  it  plays  an  important  part  in  determining  pathological  con- 
ditions of  the  true  pelvis  (such  as  salpingitis  and  metritis).  Finally  it  is  responsible 
for  most  post  mortem  and  agonic  ante  mortem  infections. 

The  colon  bacillus  is  found  in  the  soil,  in  water  contaminated  with  animal 
excreta,  and  in  dust. 


394  THE   COLON   BACILLUS 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

The  colon  bacillus  is  [usually]  pathogenic  to  guinea-pigs,  rabbits,  mice; 
and  other  animals.  Though  often  avirulent  when  isolated  from  the  stools 
of  healthy  persons,  its  virulence  can  be  rapidly  increased  by  passing  it  through 
the  peritoneal  cavities  of  a  series  of  guinea-pigs.  Guinea-pigs  are  the  most 
suitable  animals  for  the  study  of  the  experimental  disease. 

A  virulent  strain  may  easily  be  obtained  by  suturing  the  anus  of  a  guinea-pig. 
The  animal  dies  of  intestinal  obstruction,  and  a  pure  culture  of  a  very  virulent  colon 
bacillus  can  be  isolated  from  the  cloudy  peritoneal  exudate.  Should  the  exudate 
as  is  sometimes  the  case  contain  a  few  other  organisms  mixed  with  the  colon  bacillus, 
a  pure  culture  of  the  latter  can  be  readily  obtained  by  plating  on  gelatin. 

Sub-cutaneous  inoculation  of  a  colon  bacillus  of  low  virulence  into  guinea- 
pigs,  rabbits  or  mice  leads,  as  a  rule,  to  the  formation  of  an  abscess 
which  resolves  spontaneously.  Intra-peritoneal  inoculation  produces  a  more 
severe,  but  not  usually  fatal  infection. 

The  inoculation  of  a  virulent  strain,  on  the  other  hand,  usually  gives  rise 
to  an  acute  disease  in  these  animals  :  the  effects  will  be  described  in  detail. 

1.  Guinea-pigs,     (a)  Intra-peritoneal   inoculation. — The   inoculation   of  a 
few  drops  of  a  broth  culture  into  the  peritoneal  cavity  causes  death  in  about 
20  hours  with  symptoms  of  sub-acute  peritonitis  and  a  sub-normal  tem- 
perature.    Post  mortem,  there  is  a  generalized  peritonitis  with  a  copious, 
turbid  exudate  :  the  coils  of  the  intestine  are  covered  with  a  purulent  fibrinous 
exudate  :   the  lumen  of  the  gut  is  rilled  with  diarrhoeal  matter,  the  walls  are 
swollen  and  congested  and  occasionally  show  some  mucous  ecchymoses,  the 
Peyer's  patches  are  swollen  and  the  spleen  enlarged ;   in  females  the  organs 
of  generation  are  congested  and  it  is  not  uncommon  to  find  the  uterus  filled 
with   an  hsemorrhagic  exudate.     The   organism  can  be  isolated  from  the 
blood  and  internal  organs. 

(P)  Intra-pleural  inoculation. — Death  supervenes  in  24  hours.  Post 
mortem  there  is  an  excess  of  fluid,  sometimes  blood-stained,  in  the  pleura, 
with  fibrinous  deposit  on  the  lungs,  pericardial  effusion,  congestion  of  the 
lungs  and  intestine  and  swelling  of  the  spleen.  The  bacillus  is  present  in  the 
blood  and  internal  organs. 

(y)  Sub-cutaneous  inoculation. — This  leads  to  a  less  severe  infection  than 
the  preceding  and  much  larger  doses  of  culture  (1-2  c.c.)  are  required  to  pro- 
duce a  fatal  result.  A  swelling  forms  at  the  site  of  inoculation,  the  bacillus 
becomes  disseminated  and  death  takes  place  in  48  hours.  Post  mortem  the 
Peyer's  patches  and  the  spleen  are  swollen  and  the  intestine  congested  and 
ecchymosed. 

2.  Mice.— Mice,  though  less  susceptible,  succumb  to  the  inoculation  of 
cultures  of  the  bacillus.     The  lesions  are  similar  to  those  in  guinea-pigs. 

3.  Rabbits. — Eabbits  also  are  less  susceptible  than  guinea-pigs,  and  much 
larger  doses  must  be  used  to  produce  death  ;    post  mortem  the  lesions  are 
similar  in  the  two  cases. 

When  a  small  dose  is  inoculated  sub-cutaneously  the  animal  does  not  die 
for  several  days  and  post  mortem  suppurative  foci  will  be  found  in  the  liver, 
spleen  and  mesenteric  glands. 

Intra-venous  inoculation  usually  leads  to  a  rapidly  fatal  infection  ;  the 
rabbit  suffers  from  a  colon  bacillaemia  resulting  in  the  production  of  the  usual 
lesions  in  the  walls  of  the  intestine  and  spleen. 

Sometimes  the  animal  may  survive  the  intra- venous  inoculation  of  a  few  drops  of 
a  broth  culture  for  several  months.  In  such  cases  an  atrophic  paralysis  appears  as 


MORPHOLOGY  395 

the  result  of  an  anterior  poliomyelitis  (Gilbert  and  Lion) ;  this  affection  of  the  cord 
is  not  necessarily  fatal,  and  the  rabbits  sometimes  recover  even  though  the  symptoms 
may  have  been  very  marked. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  colon  bacillus,  like  the  typhoid  bacillus,  is  a  small  rod-shaped  organism 
with  rounded  ends.  Morphologically,  the  two  organisms  are  identical  and 
subject  to  the  same  variations ;  spindle-shaped  forms  and  pseudo-sporing 
forms  are  met  with  equally  in  the  two  cases. 

Staining  methods. — Like  the  typhoid  bacillus,  the  colon  bacillus  is  gram- 
negative  and  stains  with  the  ordinary  dyes. 

Mofility. — As  a  rule,  the  colon  bacillus  is  less  motile  than  the  typhoid 
bacillus. 

The  motility  varies  greatly  in  strains  from  different  sources  ;  in  some  cases  indeed 
the  bacilli  are  non-motile,  in  others  the  movements  are  slow  and  limited,  while 
in  others  again  the  organisms  are  almost  as  motile  as  the  typhoid  bacillus. 

Flagella. — The  flagella  of  the  colon  bacillus  offer  many  points  of  contrast 
with  those  of  the  typhoid  bacillus.  They  can  be  stained  by  the  same  methods 
as  the  latter  but  successful  preparations  are  more  difficult  to  obtain. 

The  number  of  flagella  is  always  smaller  than  in  the  case  of  the  typhoid 
bacillus :  the  colon  bacillus  has  usually  about  four  to  six  flagella  and  it  is 
quite  the  exception  to  find  as  many  as  twelve. 

The  flagella  may  be  arranged  all  round  the  surface  [peritrichous  ] :  but 
more  commonly  they  are  seen  arranged  in  one  or  two  bunches  attached  to 
points  on  the  surface,  generally  towards  one  end  [lophotrichous].  The 
flagella  rarely  exceed  3~5ft  in  length  being  only  an  half  to  a  third  as  long  as 
those  of  the  typhoid  bacillus  :  they  are  not  so  wavy  and  undulating  and  are 
never  seen  in  the  tangled  bunches  so  characteristic  of  the  typhoid  bacillus. 

2.  Cultural  characteristics. 

A.  Conditions  of  growth. — The  conditions  under  which  growth  takes  place 
are  the  same  for  the  typhoid  and  colon  bacilli :    both  are  able  to  grow  at 
45°  C.,  but  given  equal  opportunities  the  colon  bacillus  grows  rather  more 
quickly.     Its  growth  is  accompanied  by  an  unpleasant  faecal  odour  which  is 
characteristic  of  the  organism. 

B.  Characteristics   of  growth   on   various   media.    1.  Broth. — In  cultures 
incubated  at  37°  C.  growth  is  visible  in  6-8  hours  and  has  in  general  the  same 
characteristics  as  the  growth  of  the  typhoid  bacillus ;    a  greyish  pellicle 
however  often  forms  on  the  surface  of  the  medium  which  is  only  exceptionally 
seen  in  cultures  of  the  typhoid  bacillus. 

2.  Gelatin. — The  colon  bacillus  does  not  liquefy  gelatin. 

(a)  Stab  cultures. — In  cultures  incubated  at  20°  C.  growth  is  visible  in 
24  hours.  The  small  colonies  which  form  along  the  line  of  the  stab  become 
opaque  and  soon  unite  to  form  a  continuous  line  of  growth.  On  the  surface, 
a  thick  whitish  pellicle  of  creamy  consistence  forms  and  may  extend  to  the 
side  of  the  tube.  In  short,  the  growth  of  the  colon  bacillus  is,  as  a  rule,  both 
more  copious  and  more  rapid  than  that  of  the  typhoid  bacillus  but  the  dif- 
ferences may  not  be  very  marked  and  cannot  be  relied  upon  for  purposes  of 
differentiation. 

(/5)  Stroke  culture. — After  incubating  for  30  hours  a  thin,  bluish  layer 
with  pinked  edges  appears  which  subsequently  becomes  whitish  and  opaque. 


396  THE   COLON  BACILLUS 

In  typical  cases,  the  growth  is  more  abundant  and  more  opaque  than  that  of 
the  typhoid  bacillus. 

(y)  Isolated  colonies. — As  a  rule,  isolated  colonies  are  small  and  lenticular 
and  their  margins  indented  ;  at  first  they  are  bluish  and  transparent  but 
later  become  white  and  opaque  and  are  larger  than  those  of  the  typhoid 
bacillus.  Frequently,  however,  the  colonies  remain  transparent  and  preserve 
the  "  iceberg  "  appearance  already  noted  as  characteristic  of  the  typhoid 
bacillus. 

Colonies  which  develop  in  the  depth  of  the  gelatin  have  the  appearance 
of  small  whitish  opaque  grains. 

3.  Agar  and  coagulated  serum. — On  these  media  the  colon  bacillus  forms  a 
whitish  layer  with  no  characteristic  feature.     Gas-bubbles  sometimes  form 
in  the  depth  of  the  medium  and  increasing  in  size  lift  up  the  medium. 

4.  Potato. — As  a  rule,  the  growth  is  at  first  yellowish  and  then  later  becomes 
brown,  thick,  raised  and  moist ;    but   some   strains   of  the   colon   bacillus 
give  a  thin  colourless  pellicle  indistinguishable  from  the  growth  of  the  typhoid 
bacillus.     The  quality  and  variety  of  the  potato  used  have  much  to  do  with 
the  appearance  of  the  growth. 

On  Remy  and  Sugg's  solid  medium  the  colon  bacillus  invariably  gives  rise  to  an 
abundant,  thick  growth  which  may  be  glairy  or  dry  and  which  is  always  of  a  dirty 
yellow  or  brown  colour. 

5.  Milk. — Milk  is  coagulated  in  24-30  hours  when  incubated  at  37°  C. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Biochemical  reactions. 

1.  Action  on  carbohydrates. — In  both  aerobic  and  anaerobic  culture  the 
colon  bacillus  decomposes  laevulose,  lactose,  saccharose,  maltose,  glucose, 
erythrite,  and  mannite,  with  the  formation  of  acid  (formic,  acetic,  butyric, 
lactic),  gas  (hydrogen,  carbon-dioxide),  and  ethyl  alcohol.  These  reactions 
are  invaluable  for  the  purpose  of  identifying  the  organism.  The  technique 
has  been  described  in  connexion  with  the  action  of  the  typhoid  bacillus  on 
sugars  (p.  373). 

Attention  must  be  drawn  to  the  fact  that  the  colon  bacillus  under  certain  condi- 
tions, particularly  when  it  is  grown  in  symbiosis  with  the  typhoid  bacillus  (Remy), 
may  lose  its  power  of  splitting  up  sugars  with  the  formation  of  acid  and  gas.  Grim- 
bert  and  Legros  have  found  however,  that  in  some  cases  where  dysgonic  influences 
have  affected  the  fermentation  properties  of  the  colon  bacillus  these  properties  though 
markedly  diminished,  are  not  altogether  lost ;  they  have  been  able  to  show,  for 
instance,  that  milk  will  be  coagulated  if  in  a  shallow  layer,  and  that  lactose  if  present 
in  sufficient  quantity  is  feebly,  but  nevertheless  definitely  attacked. 

(a)  Action  on  lactose-broth  containing  calcium  carbonate. — When  sown 
on  this  medium  and  incubated  at  37°  C.  for  12-20  hours  the  colon  bacillus 
decomposes  the  lactose  with  the  formation  of  acids,  which  in  turn  attack  the 
calcium  carbonate  and  give  rise  to  numerous  bubbles  of  carbon-dioxide. 

(fc)  Action  on  litmus  in  presence  of  a  carbohydrate  fermented  by  the 
organism. — Litmus-lactose-gelatin  and  litmus-mannite-gelatin.  The  blue 
colour  of  the  litmus  is  first  changed  to  red  and  later  assumes  a  peculiar  colour 
somewhat  resembling  that  of  the  skin  of  an  onion. 

(c)  Action  on  Grimbert  and  Legros'  medium  (p.  373). — The  colour  of  the 
medium  is  rapidly  changed  to  red. 

(d)  Milk. — Milk  is  rapidly  coagulated  (vide  p.  373). 

[(e)  Litmus  milk.— The  litmus  is  first  turned  pink  and  subsequently 
bleached  ] 


m  mA     B    4ft 


BIOLOGICAL  PROPERTIES  397 

Numerous  more  or  less  ingenious  methods  have  been  devised  to  illustrate  the 
fermentation  properties  of  the  colon  bacillus.  Thus  for  instance,  to  an  agar  or  other 
medium  containing  lactose,  a  substance  (e.g.  fluorescin)  is  added  which  is  altered  or 
intensified  in  colour  by  the  acids  formed  out  of  the  lactose  :  in  other  cases  a  reagent 
is  selected  for  addition  to  the  medium  which  is  coloured  in  alkaline  solutions  but 
colourless  in  acid  solutions  (e.g.  phenol-phthalein).  Ramond's  method  may  be 
described  as  an  example. 

(/)  Ramond's  method. — Take  a  tube  of  gelatin  containing  4  per  cent,  of  lactose 
and  after  melting  it — being  careful  not  to  apply  too  much  heat — add  sufficient 
aqueous  solution  of  acid  fuchsin  (Rubin  8.)  to 
impart    a    red-cerise    colour    to    the    gelatin, 
then  just  decolourize  with  a  saturated  aqueous 
solution  of  sodium  carbonate — 2—3  drops  are 
sufficient — filter,    sterilize    at    105°  C.    for   5 
minutes  and  pour  the  now  colourless  medium 
into  a  sterile  Petri  dish.     The  typhoid  bacillus 
produces  no  change  of  colour  when  sown  on 
this    medium,  while   on   the   other  hand   the 
colon  bacillus,  in  virtue  of  the  acids   formed 
from  the  lactose  which  neutralize  the  sodium    ffi  when  grown  on  Ramond's  agar.    (After 
carbonate,  regenerates  the  red  tint  so  that  a    Gauthie\) 
characteristic    rose  -  coloured    area    develops 

around  colonies  of  this  organism.     This  method  is  not  so  delicate  as  that  with 
litmus- tinted  media. 

2.  Action  on  neutral  red. — Neutral  red  in  culture  media  is  reduced  and 
decolourized  by  the  colon  bacillus.     The  typhoid  bacillus  has  no  action  on 
the  dye. 

Liquefy  a  tube  containing  10  c.c.  of  ordinary  agar  (or  glucose-agar),  add  3  or  4 
drops  of  a  sterile  saturated  aqueous  solution  of  neutral  red,  and  when  the  medium 
has  cooled  and  set  sow  it  with  the  colon  bacillus  in  stab  culture  and  incubate  at 
37°  C.  for  24  hours.  The  medium  will  now  no  longer  be  red  but  will  exhibit  a 
greenish  fluorescence,  and  on  further  incubation  this  will  soon  change  to  a  canary 
yellow  colour.  This  reaction  has  been  adapted  by  Savage  to  the  detection  of  the 
colon  bacillus  in  water  (p.  411). 

3.  Indol  formation. — An  important  and  very  constant  characteristic  of  the 
colon  bacillus  is  the  formation  of  indol  in  culture  media. 

The  value  of  the  indol  reaction  in  the  diagnosis  of  the  colon  bacillus  has  been 
called  in  question  by  some  authors  on  the  ground  that  they  not  infrequently  fail  to 
find  any  indol  in  cultures  of  this  organism  :  and  Remy  has  shown  that  when  the 
colon  bacillus  is  grown  with  the  typhoid  bacillus  the  former  may  lose  its  capacity 
to  produce  indol. 

Recent  work  demonstrates  that  the  negative  results  obtained  by  the  earlier 
observers  were  due  to  the  imperfections  of  their  technique.  "  The  property  of 
producing  indol  is  far  less  variable  than  is  generally  believed,"  and  the  indol  reaction 
furnishes  one  of  the  best  tests  there  is  for  identifying  the  organism,  provided  that 
the  following  precautions  be  observed,  viz.  : — 

1.  That  peptone  water  and  not  ordinary  broth  be  used  as  the  culture  medium. 

2.  That  the  culture  be  examined  between  the  third  and  the  eighth  day  but  never 
later. 

3.  That  the  test  be  performed  exactly  as  described  at  p.  374. 

4.  Cultures   on   synthetic   media. — The   colon   bacillus   as    a    rule    grows 
luxuriantly  in  the  different  liquid  media  of  Nsegeli,  Maasse,  Frankel,  Remy 
and  Sugg '(p.  375). 

5.  Growth  on  vaccinated  media. — If  the  colon  bacillus  be  sown  on  a  tube 
of  agar  or  gelatin  on  which  the  typhoid  bacillus  has  already  been  grown  and 
scraped  off  as  described  above,  some  amount  of  growth  generally  takes  place 
which  though  distinct  is  less  abundant  than  on  tubes  of  new  media. 

6.  Growth    on    coloured    media. — The    colon    bacillus    decolourizes    both 


398  THE   COLON   BACILLUS 

Nceggerath's  medium  and  fuchsin-agar.     The  typhoid  bacillus  gives  similar 
results. 

7.  Growth  on  arseniated  broth. — A  typical  colon  bacillus  grows  in  broth 
containing  as  much  as  2  grams  of  arsenious  acid  per  litre  (Thoinot  and 
Brouardel). 

8.  Growth  on  artichoke. — A  typical  colon  bacillus  grows  luxuriantly  on 
artichoke,  and  turns  the  medium  green  (p.  375). 

9.  Growth  on  media  containing  caffeine.— The  colon  bacillus  does  not 
grow  on  media  containing  0*5  per  cent,  of  caffeine  (p.  408). 

10.  Growth  on  malachite-green  media. — According  to  Loeffler  the  addition 
of  a  small  quantity  of  malachite-green  to  culture  media  prevents  the  growth 
of  the  colon  bacillus,  but  does  not  interfere  with  the  growth  of  the  typhoid 
bacillus.     As  a  matter  of  fact,  the  colon  bacillus  grows  on  media  containing 
either  malachite  green  or  crystal  violet  (pp.  409  and  407). 

2.  Variability  of  flagella. 

The  variability  of  the  flagella  is  very  limited,  their  characteristics  being 
little  influenced  by  antiseptics,  temperatures  unfavourable  to  growth,  etc. 
(Kemy  and  Sugg). 

Examination  of  the  flagella  should  never  be  neglected  when  it  is  desired  to 
identify  the  colon  bacillus. 

3.  Vitality  and  Virulence. 

Vitality. — All  that  has  been  said  with  regard  to  the  vitality  of  the  typhoid 
bacillus  is  equally  applicable  to  the  colon  bacillus. 

Virulence. — The  virulence  of  the  colon  bacillus  is  subject  to  great  variation 
(vide  experimental  inoculation,  p.  394). 

4.  Toxin. 

Malvos  has  shown  that  porcelain-filtered  broth  cultures  are  toxic.  Broth 
cultures  also  yield  a  toxic  precipitate  when  heated  with  sulphate  of  ammonia. 
As  a  rule,  the  toxin  is  not  very  harmful  and  large  doses  of  filtered  cultures 
must  be  inoculated  to  produce  a  fatal  result  in  experimental  animals. 

The  inoculation  of  a  large  dose  of  toxin  into  the  ear- vein  of  a  rabbit  pro- 
duces the  following  symptoms  :  At  first  there  is  muscular  weakness,  sub -normal 
temperature,  drowsiness  and  coma  :  later,  convulsions  set  in  and  finally  a 
generalized  tetanic  condition  which  continues  till  the  animal  dies  (Gilbert). 
A  smaller  dose  produces  a  chronic  intoxication  with  diarrhoea,  drowsiness 
and  wasting,  the  animal  often  dying  of  cachexia. 

In  guinea-pigs,  the  inoculation  of  large  quantities  of  toxin  into  the  peri- 
toneal cavity  is  followed  by  a  sub-normal  temperature  and  leads  to  collapse 
and  death  (Boix).  The  blood  may  contain  organisms  (especially  the  colon 
bacillus)  which  have  found  their  way  from  the  intestinal  canal  (Achard  and 
Renault). 

Colilysin. — In  suitable  media  the  colon  bacillus  forms  an  haemolytic  sub- 
stance (Kayser). 

Colilysin  is  only  produced  in  any  quantity  if  the  broth  has  a  markedly  acid 
reaction  (80  c.c.  of  decinormal  oxalic  acid  per  litre). 

The  hsemolysin  is  present  after  incubating  for  2  days  at  37°  C.  but  continues  to 
increase  in  amount  until  the  fourth  day  and  remains  at  its  maximum  until  the  end 
of  the  second  week. 

Colilysin  is  a  powerful  solvent  of  dog  red-cells  ;  it  has  less  action  on  horse, 
ox,  and  rabbit  cells,  and  very  little  and  in  some  cases  no  action  at  all  on  the 


AGGLUTINATION  399 

red  cells  of  other  animals  (man,  guinea-pigs,  birds  etc.).  Colilysin  can  be 
kept  for  months  at  the  ordinary  temperature  of  the  laboratory  and  is  not 
destroyed  by  heating  to  120°  C.  for  half  an  hour. 

Some  normal  serums  (those  of  man,  the  horse  etc.)  neutralize  the  hsemolytic 
property  of  colilysin :  and  an  anti-colilysin  can  be  readily  produced  by  inoculating 
various  animals  sub-cutaneously  with  four-day  old  broth  cultures  of  the  colon 
bacillus. 

5.  Vaccination  and  serum  therapy. 

Guinea-pigs  and  rabbits  can  be  immunized  by  repeatedly  inoculating  them 
either  with  small  doses  of  living  and  virulent  organisms  or  with  filtered 
cultures  of  similar  strains.  Albarran  and  Mosny  produced  a  very  high  degree 
of  immunity  in  dogs  and  rabbits  by  repeatedly  inoculating  them  with  small 
doses  of  filtered  cultures  and  with  the  filtrates  derived  from  macerating  the 
internal  organs  of  animals  dead  of  a  colon  bacillus  infection.  Rodet  im- 
munized horses  and  sheep  by  inoculating  them  repeatedly  with  increasing 
doses  of  living  or  dead  cultures. 

The  serum  of  vaccinated  animals  has  marked  immunizing  properties  and 
also,  to  some  extent,  therapeutic  properties.  These  properties  are  mani- 
fested against  the  strains  used  for  immunization  but  may  be  wanting  against 
strains  from  other  sources. 

Antityphoid  serum  is  neither  prophylactic  nor  curative  for  the  colon 
bacillus. 

According  to  the  experiments  of  Sanarelli  and  some  other  observers  animals 
vaccinated  against  the  colon  bacillus  should  be  immune  to  both  the  colon  and  typhoid 
bacilli,  and  the  serum  of  the  animals  should  immunize  against  the  typhoid  bacillus. 
These  results  have  however  not  been  confirmed. 

6.  Agglutination. 

(a)  The  serum  of  animals  infected  with  the  colon  bacillus  or  immunized 
against  that  organism,  as  well  as  the  serum  of  persons  suffering  from  infections 
due  to  the  colon  bacillus,  have  the  property  of  agglutinating  the  bacillus. 
The  agglutination  reaction  is  always  obtained  with  the  strain  producing  the 
infection,  but  the  results  are  often  negative  if  other  than  the  infecting  organism 
be  employed  for  the  reaction,  though  the  latter  may  be  an  authentic  colon 
bacillus.     This  method  of  diagnosis  cannot  therefore  be  relied  upon.     The 
capacity  of  the  colon  bacillus  to  agglutinate  is  increased  to  a  very  marked 
extent  by  sub-culturing  it  on  artificial  media  (Rodet). 

(b)  The  colon  bacillus  is  not  agglutinated  by  the  serum  of  animals  vaccinated 
against  the  typhoid  bacillus  nor  by  the  serum  of  persons  suffering  from  enteric 
fever.     But  for  this  reaction  to  be  of  any  value  it  is  important  that  certain 
precautions  be  observed  (vide  footnote  on  p.  389). 

All  human  serums  whether  taken  from  enteric  fever  patients  or  not  exert 
a  slight  agglutinating  action  on  the  colon  bacillus  when  diluted  five  or  ten 
times.  Unless  this  fact  be  borne  in  mind  it  may  lead  to  error.  All  mistakes 
may  be  avoided  by  adopting  the  following  methods. 

Determine  carefully  first  of  all  the  agglutinating  power  of  the  typhoid 
serum  which  is  to  be  used  in  the  reaction  :  then  mix  a  drop  of  the  highest 
dilution  of  the  serum  which  will  definitely  agglutinate  the  typhoid  bacillus 
with  a  culture  of  the  colon  bacillus.  Thus,  for  example,  if  the  highest  dilution 
in  which  a  given  typhoid  serum  will  agglutinate  the  typhoid  bacillus  be 
1-100  this  dilution  of  the  serum  should  be  used  in  testing  the  suspected  colon 
bacillus.  Under  these  conditions  the  agglutination  of  the  colon  bacillus  is 
never  observed,  and  the  serum  reaction  can  be  employed  as  an  excellent 
means  for  differentiating  the  two  organisms  provided  that  it  be  always  remem- 


400  THE  COLON  BACILLUS 

bered  that  a  strain  of  the  typhoid  bacillus  which  is  not  agglutinated  by  a 
typhoid  serum  may  very  occasionally  be  encountered. 

SECTION  IV.— DETECTION,   ISOLATION  AND   IDENTIFICATION. 

The  methods  of  detecting  the  colon  bacillus  in  the  tissues  and  fluids  of  the 
body  are  similar  in  principle  to  those  employed  for  the  detection  of  the  typhoid 
bacillus.  These  methods  as  well  as  the  differentiating  tests,  etc.  are  fully 
dealt  with  in  Chap.  XXIII. 

It  must  be  remembered  that  the  colon  bacillus  often  multiplies  in  the 
body  immediately  after  death,  and  even  during  the  last  few  hours  of  life  : 
the  finding  of  the  colon  bacillus  in  the  tissues  or  fluids  under  these  conditions 
is  therefore  of  no  diagnostic  value  whatever. 

The  bacillus  of  Green  Diarrhoea. 

According  to  Lesage  and  Thiercelin  the  bacillus  of  green  diarrhoea  is  merely  a 
chromogenic  variety  of  the  colon  bacillus.  The  organism  is  found  in  practically 
pure  culture  in  the  stools  of  children  suffering  from  the  disease. 

Experimental  inoculation. — The  organism  is  only  slightly  pathogenic  for  laboratory 
animals.  Rabbits,  when  inoculated  intra-venously  or  fed  with  cultures  of  the  bacillus, 
suffer  from  an  attack  of  green  diarrhoea  from  which  they  recover  in  a  few  days. 

Microscopical  appearance. — Morphologically  the  bacillus  is  a  short  rod-shaped 
organism  with  rounded  ends  in  every  way  similar  to  the  colon  bacillus. 

Cultures. — The  bacillus  of  green  diarrhoea  is  a  facultative  aerobe.  It  grows  on 
all  the  ordinary  media  and  gives  rise  to  a  disagreeable  odour.  The  green  colouring 
matter  is  only  produced  in  aerobic  culture. 

A  pure  culture  is  very  easily  obtained  by  plating  a  trace  of  the  stool  of  an  infected 
child  on  gelatin. 

Broth. — At  first  the  medium  is  uniformly  cloudy  but  later  a  greenish  sediment 
is  deposited. 

Gelatin  is  not  liquefied.  In  stab  culture,  the  bacillus  gives  rise  to  a  scanty  whitish 
growth  in  the  substance  of  this  medium  and  on  the  surface  to  a  smalf  greenish 
lenticular  pellicle.  On  sloped  gelatin,  the  growth  is  poor,  greenish  in  colour  and  has 
a  tendency  to  spread  away  from  the  line  of  sowing :  after  a  few  days  the  gelatin  is 
tinted  uniformly  green.  Isolated  colonies  form  small  greenish  granular  points. 

On  agar. — The  growth  is  poor,  greenish  in  colour  and  spreading.  The  agar  acquires 
a  green  tint. 

On  potato. — The  growth  is  luxuriant,  covers  the  whole  surface  of  the  medium 
and  is  of  a  dirty  green  mucous  appearance. 

M ilk  is  rapidly  coagulated. 

Carbohydrate  media  are  strongly  fermented. 


CHAPTEE  XXIII. 

THE  ISOLATION  OF  THE-TYPHOID  AND  COLON  BACILLI 
FROM  WATER,  STOOLS,  ETC.  AND  THE  METHODS 
OF  IDENTIFYING  THE  TWO  ORGANISMS. 

Introduction. 

Section  I. — The  isolation  of  the  typhoid  and  colon  bacilli,  p.  402. 

1.  Original  methods,  p.  402.  2.  Eisner's  method  and  its  modifications,  p.  403. 
3.  Precipitation  methods,  p.  406.  4.  Method  based  upon  the  motility  of  the  typhoid 
bacillus,  p.  406.  5.  Chantemesse's  carbolic  media,  p.  407.  6.  Conradi-Drigalski's 
method,  p.  407.  7.  Endo's  medium,  p.  408.  8.  Caffeine  media,  p.  408.  9.  Malachite 
green  media,  p.  409.  10.  China  green  medium,  p.  410.  11.  Bile  media,  p.  410.  12. 
Brilliant  green  medium,  p.  411.  13.  Neutral  red  media,  p.  411.  14.  Methods  based 
upon  agglutination,  p.  412.  15.  MacConkey's  media,  p.  412. 

Section  II. — The  identification  of  the  typhoid  and  colon  bacilli,  p.  412. 

THE  isolation  of  the  typhoid  bacillus  from  water,  etc.  in  which  it  is  mixed 
with  other  species  of  organisms,  and  especially  when  the  colon  bacillus  is 
also  present,  presents  certain  difficulties  which  may  be  summed  up  under 
four  headings. 

1.  On  gelatin  media,  at  the  ordinary  temperature  of  the   atmosphere, 
colonies  of  the  typhoid  bacillus  develop  slowly  (requiring  about  48  hours) 
while  saprophytic  organisms  which  liquefy  the  medium  grow  more  quickly 
and  so  put  an  end  to  the  investigation. 

2.  The  colon  bacillus  very  often  retards  the  growth  of  the  typhoid  bacillus 
when  the  two  organisms  are  sown  together  on  artificial  culture  media,  with 
the  result  that  the  presence  of  the  latter  may  pass  unnoticed.     There  is,  in 
fact,  a  true  antagonism  between  the  colon  bacillus  and  the  typhoid  bacillus 
(Grimbert).     A  similar  antagonism  also  exists  between  certain  other  micro- 
organic  species  and  the  typhoid  bacillus  when  sown  together  on  artificial 
media  (Besson). 

3.  Remy,  though  he  does  not  admit  that  the  typhoid  bacillus  is  crowded 
out  by  the  colon  bacillus,  nevertheless  lays  stress  on  the  difficulty  of  isolating 
the  former  when  the  latter  organism  is  also  present.     He  shows  that  by 
growing  the  two  organisms  together  their  properties  may  be  profoundly 
modified  :    thus  the  typhoid  bacillus  occasionally  loses  its  property  of  being 
agglutinated  by  a  specific  serum,  and  the  colon  bacillus  may  under  like  con- 
ditions lose  its  indol-producing  and  fermentation  properties. 

4.  The  ordinary  method  of  gelatin-plating  only  permits  of  a  very  small 
quantity  of  a  suspected  water  being  sown  and  it  is  therefore  possible  that  if 
the  typhoid  bacillus  be  present  only  in  small  numbers  as  compared  with  other 
organisms,  it  may  escape  notice. 

2c 


402  ISOLATION   OF  THE   TYPHOID   BACILLUS 

It  is  not  a  matter  for  surprise  therefore  to  find  that  much  experimental 
work  has  been  done  with  a  view  to  perfecting  a  method  or  methods  of  detecting 
with  certainty  the  presence  of  the  typhoid  bacillus  in  material  in  which  it 
may  be  suspected  to  occur. 


SECTION  I.— THE  ISOLATION  OF  THE  TYPHOID   AND   COLON 

BACILLI. 
1.  Original  methods. 

Under  this  heading  will  be  briefly  considered  various  methods  which  though 
in  use  until  recently  do  not  give  dependable  results,  being  practically  useless 
for  detecting  the  typhoid  bacillus  when  the  latter  is  mixed  with  the  colon 
bacillus.  These  methods  are  now  almost  entirely  discarded. 

(a)  Rodet's  method. — Rodet  showed  that  the  typhoid  and  colon  bacilli  would  grow 
at  45°  C.  while  most  other  organisms  failed  to  do  so,  and  on  this  fact  based  the  following 
method  of  analysis.  To  a  flask  containing  sterilized  broth  he  added  20-100  c.c.  of  the 
suspected  water  and  incubated  at  45°  C.  for  20-24  hours.  If  on  taking  the  flask  out  of 
the  incubator  the  broth  was  cloudy  a  strong  presumption  was  raised  that  the  typhoid 
or  colon  bacillus  or  both  were  present  in  the  water.  Microscopical  examination  of  the 
culture  and,  if  need  be,  isolation  on  gelatin  plates  removed  all  doubt. 

(6)  Method  of  Chantemesse  and  Widal. — Chantemesse  and  Widal  found  that  both 
typhoid  and  colon  bacilli  would  grow  in  artificial  media  containing  2'5  grams  of  carbolic 
acid  per  litre,  and  utilized  the  fact  in  order  to  detect  these  organisms  in  water. 

To  tubes  containing  20  c.c.  of  liquefied  gelatin  add  1  c.c.  of  a  5  per  cent,  solution  of 
carbolic  acid  and  a  few  drops  of  the  water  to  be  examined  and  pour  plates.  Unfortunately 
a  certain  number  of  organisms  develop  in  the  plates  which,  as  they  grow,  liquefy  the 
medium  and  consequently  soon  put  an  end  to  the  experiment.  A  large  number  of  plates 
must  be  sown  with  each  of  the  suspected  samples  because  only  a  very  small  amount  of 
water  can  be  used  for  each  plate. 

(c)  Vincent's  method. — Vincent  devised  a  method,  which  for  a  long  time  was  in  general 
use,  based  upon  a  combination  of  the  two  preceding  observations.     He  used  broth  con- 
taining O'l  per  cent,  of  carbolic  acid  as  the  culture  medium  and  incubated  the  cultures 
at41-5°-42°C. 

To  each  of  half-a-dozen  tubes  containing  10  c.c.  of  broth  add,  immediately  before  use, 
5  drops  of  a  5  per  cent,  solution  of  carbolic  acid.  Sow  with  O'5-l  c.c.  of  the  suspected 
water,  cover  with  india-rubber  caps  to  prevent  evaporation  of  the  carbolic  acid,  and 
incubate  at  41 '5°  or  42°  C.  If  the  medium  in  any  of  the  tubes  becomes  cloudy  after 
incubating  for  12  or  20  hours,  transfer  a  little  of  the  culture  to  a  fresh  tube  of  carbolic- 
broth  and  incubate  it  similarly  at  41 '5°  C.  As  a  rule,  when  the  suspected  water  contains 
the  colon  bacillus  the  first  sub-culture  yields  a  pure  growth  of  the  latter  organism.  It 
must,  however,  be  borne  in  mind  that  some  saprophytes  (Bacillus  subtilis,  Bacillus  mesen- 
tericus,  B.  luteus,  the  white  streptococcus  of  water,  Proteus  vulgaris,  etc.)  will  also  grow 
under  these  conditions.  These  latter  organisms  cannot  be  excluded  by  further  sub- 
cultivation  in  carbolic -broth  because  once  they  become  accustomed  to  carbolic  media 
they  grow  in  them  just  as  well  as  the  colon  bacillus.  A  watered  silk  appearance  in  the 
tubes  is  a  fairly  reliable  indication  of  the  presence  of  the  colon  or  typhoid  bacillus,  but 
the  investigation  must  always  be  carried  further  by  microscopical  examination  and 
isolation  on  gelatin.  It  is  well  to  remember  that  in  carbolic-broth  the  colon  and  typhoid 
bacilli  often  occur  as  very  short  rods  (cocco-bacilli)  arranged  in  pairs  and  devoid  of 
motility. 

(d)  Method  of  P6re". — This  is  merely  Vincent's  method  modified  in  such  a  way  as  to 
allow  large  quantities  of  the  suspected  water  to  be  examined. 

Prepare  a  concentrated  broth  (meat,  1000  grams,,  water  1000  grams,  and  peptone 
50  grams),  distribute  in  quantities  of  50  c.c.  in  a  series  of  flasks,  and  autoclave. 

To  each  flask  add  3  c.c.  of  a  5  per  cent,  solution  of  carbolic  acid  and  100  c.c.  of  the 
suspected  water.  Sow  five  or  six  flasks  and  incubate  them  at  41°  C.  As  soon  as  the 
medium  becomes  cloudy  (15-20  hours)  sow  a  series  of  broth  tubes  each  containing  O'l  per 
cent,  carbolic  acid  with  a  trace  of  the  growth  from  any  of  the  flasks  that  may  be  cloudy. 
Incubate  at  41°  C.  and  continue  the  experiment  as  in  Vincent's  method. 

(e)  Method  of  Pouchet  and  Bonjean. — This  also  is  a  modification  of  Vincent's  method. 
To  each  of  a  series  of  flasks  containing  100  c.c.  of  sterile  broth  add  150  c.c.  of  the  water 
to  be  examined  and  5  c.c.  of  a  5  per  cent,  solution  of  carbolic  acid.     Incubate  at  42°  C. 


ELSNER'S   METHOD  403 

If  the  medium  becomes  cloudy  in  any  of  the  flasks  sow  sub-cultures  for  three  generations 
in  O'l  per  cent,  carbolic  acid  broth  and  incubate  at  42°  C.  Finally,  sow  a  tube  of  ordinary 
broth  from  the  last  carbolic  broth  culture,  incubate  at  36°  C.  for  8  days  and  then  inoculate 
a  guinea-pig  with  0*3  c.c.  of  culture  per  100  grams  of  animal.  If  the  animal  die  sow 
cultures  with  fragments  of  the  internal  organs  and  heart  blood. 

The  five  methods  just  described  are  available  for  the  isolation  of  the  typhoid  bacillus 
provided  that  the  colon  bacillus  is  not  also  present  but  if,  as  is  most  often  the  case,  the 
two  organisms  are  present  together  the  isolation  of  the  former  is  impossible  by  these 
means. 

2.  Eisner's  method  and  its  modifications. 
A.  Eisner's  method. 

The  method  is  available  according  to  Eisner  for  the  isolation  of  the  typhoid 
bacillus  from  sources  such  as  water  or  stools  in  which  the  colon  bacillus  is 
also  present. 

The  technique  is  based  upon  the  fact  that  the  typhoid  and  the  colon  bacilli 
grow,  to  the  exclusion  of  most  other  organisms,  on  a  potato-jelly  containing 
iodide  of  potassium.  Disappointing  results  are  however  frequently  obtained  ; 
sometimes  the  plates  are  rapidly  liquefied  and  the  experiment  brought  to  an 
end  ;  at  other  times  the  typhoid  bacillus  cannot  be  found  even  though  it 
has  been  purposely  introduced  into  a  sample  of  water  as  a  control.  Several 
attempts  have  been  made  to  improve  the  method,  and  these  will  be  considered 
subsequently. 

Technique.     A.  Isolation  from  water. — 1.  Prepare  and  sterilize : — (i)  a  number 
of  tubes  each  containing  10  c.c.  of  potato  gelatin  (p.  41). 
(ii)  The  following  solution  : — 

Distilled  water,  50  grams. 

Potassium  iodide,      -  10        „ 

2.  Immediately  before  use,  melt  the  potato-gelatin  tubes  and  add  1  c.c.  (20  drops) 
of  the  iodide  solution. 

The  gelatin  will  then  contain  1  per  cent,  of  iodide. 

3.  Sow  ten  to  fifteen  tubes  each  with  0*5  or  1  c.c.  of  the  suspected  water  and 
plate. 

4.  According  to  Eisner,  the  colon  bacillus  appears  on  these  plates  as  early  as  the 
second  day  (at  22°  C.)  as  circular,  opaque,  slightly  brown  colonies  while  the  typhoid 
bacillus  does  not  develop  until  the  plates  have  been  incubated  for  4  days  and  then 
as  smaller,  transparent,  barely  visible  colonies.     Other  organisms  fail  to  grow. 

As  a  matter  of  fact,  various  organisms  other  than  the  typhoid  and  colon  bacilli, 
and  some  of  which  liquefy  the  gelatin,  do  grow  on  the  medium ;  and  then  again  the 
colonies  of  the  typhoid  bacillus  are  not  so  easily  differentiated  as  Eisner  makes  out. 
It  must  be  distinctly  realized  that  Eisner's  medium  possesses  no  specific  property 
which  ensures  the  development  of  the  typhoid  and  colon  bacilli  to  the  exclusion  of 
other  organisms.  Its  only  advantage  is  that  it  allows  the  typhoid  bacillus  an  equal 
opportunity  with  the  colon  bacillus  to  grow.  It  is  necessary,  therefore,  to  examine 
carefully  every  colony  on  the  plates  which  does  not  liquefy  the  medium  and  which 
does  not  form  pigment.  This  is  easily  done  by  transferring  them  each  to  a  separate 
tube  of  broth  and  then  incubating  at  37°  C.  After  incubating  for  24  hours  the 
morphology  of  the  organisms  is  determined  by  examining  the  cultures  microscopically 
and  only  those  tubes  which  show  short,  gram-negative  bacilli  with  rounded  ends 
need  be  reserved  for  the  further  tests  to  be  described  later. 

If  any  of  the  broth  cultures  prove  to  be  impure  they  must  be  plated  out  again  on 
Eisner's  jelly.  Sow  a  loopful  of  the  broth  in  a  fresh  tube  of  the  jelly,  a  drop  of  this 
on  a  second  tube,  and  three  drops  of  the  second  into  a  third  tube  (p.  77). 

B.  Isolation  from  stools. — The  technique  to  be  adopted  in  this  case  is  similar  to 
that  just  described.  Dilute  a  loopful  of  the  stool  in  a  tube  of  sterile  water  and  use 
a  drop  of  the  dilution  to  sow  a  tube  of  Eisner's  gelatin  :  mix  thoroughly  and  transfer 
a  drop  to  a  second  tube  and  from  the  second  tube  two  or  three  drops  to  a  third  tube. 
Pour  plates  and  incubate.  All  the  non-liquefying  colonies  which  develop  must  be 
picked  off  for  further  investigation  in  the  manner  described  above. 


404  ISOLATION   OF   THE   TYPHOID   BACILLUS 

B.  Grimbert's  method. 

Grimbert  attributes  the  failure  of  Eisner's  method  partly  to  the  want 
of  uniformity  of  the  medium  due  to  variations  in  the  chemical  composition 
of  potatoes,  and  partly  to  the  fact  that  Eisner  did  not  test  the  reaction  of 
his  medium.  According  to  Grimbert  the  addition  of  iodide  of  potassium  is 
not  essential :  ordinary  gelatin  can  be  used  if  the  reaction  be  such  that 
10  c.c.  are  neutralized  by  5  c.c.  of  lime  water,  though  it  is  better  to  have  a 
medium  of  constant  chemical  composition.  Grimbert's  medium  is  used  in 
the  same  way  as  Eisner's,  but  the  colonies  are  more  slow  in  developing  and 
the  earliest  do  not  appear  before  the  third  day.  The  method,  as  a  matter  of 
fact,  has  hardly  any  advantage  over  Eisner's  original  method. 
Technique. — To  1,000  c.c.  of  water  add  :— 

Maltose,  -  1  gram. 

Soluble  starch,  2  grams. 

Asparagin,        -  2         „ 

Neutral  phosphate  of  potassium.  2         ,, 

Potassium  sulphate,  -  2 

Magnesium  sulphate,  2         ,, 

Ammonium  bimalate,         -  2         ,. 

Magnesium  carbonate,        -  1  gram. 

Dissolve  15  per  cent,  of  gelatin  in  the  mixture,  clear  with  white  of  egg,  heat  to 
115°,  filter,  and  test  the  reaction  thus :  dilute  10  c.c.  of  the  gelatin  with  50  c.c.  of 
warm  distilled  water,  add  a  few  drops  of  an  alcoholic  solution  of  phenol-phthalein, 
then  run  in  lime  water  until  a  permanent  rose  pink  colour  is  obtained.  If  more 
than  3  c.c.  of  lime  water  are  required  to  neutralize  the  gelatin  reduce  the  acidity  by 
the  addition  of  a  small  quantity  of  normal  soda  solution  until  10  c.c.  of  the  gelatin 
are  neutralized  with  5  c.c.  of  lime  water. 

Immediately  before  use  1  per  cent,  of  iodide  or  bromide  of  potassium  may  be 
added. 

C.  Remy's  method. 

Kemy  suggests  the  use  of  a  medium  which  is  more  nutritive  and  less  acid 
than  Grimbert's.  By  means  of  his  "  differential  gelatin  "  he  has  been  able 
to  isolate  the  typhoid  bacillus  from  stools  in  all  the  cases  of  enteric  fever 
which  he  has  investigated. 

This  "  differential  gelatin  "  has  no  greater  selective  property  than  Eisner's 
medium  and  the  majority  of  micro-organisms  grow  in  it.  Still,  liquefying 
species  are  to  some  extent  checked  and  the  inhibiting  influence  of  the  colon 
bacillus  on  the  typhoid  bacillus  is  not  apparent  on  this  medium. 

Technique. — Preparation  of  the  "  differential  gelatin."     To  a  litre  of  water  in  a 
flask  add : 

Asparagin,  -         6       grams. 

Oxalic  acid,      -  -         0'5    gram. 

Lactic  acid,      -  -         0'15      „ 

Citric  acid,        -          -  ....         Q*15 

Di- sodium  phosphate,         -  -         5       grams. 

Potassium  sulphate,  -  -         -          -         1*25     ,, 

Sodium  chloride,       -         -         -         -         -         -         -         -         2 

Witte's  peptone,        -  ....       30 

Heat  to  110°  C.  for  15  minutes,  and  on  taking  the  flask  out  of  the  autoclave  pour 
the  boiling  liquid  into  another  flask  containing  120-150  grams  of  best  quality  gelatin. 
Shake  the  flask  until  the  gelatin  is  dissolved,  add  soda  solution  until  the  mixture  is 
slightly  alkaline,  heat  in  the  autoclave  again  to  110°  C.  for  15  minutes,  then  add 
sufficient  half-normal  sulphuric  acid  x  to  render  the  medium  acid  to  such  an  extent 
that  10  c.c.  require  the  addition  of  0'2  c.c.  of  half-normal  solution  of  soda  to  neutralize ; 2 
mix  by  shaking  well,  then  heat  in  the  steamer  at  100°  C.  for  10  minutes  and  filter. 

1  A  normal  solution  contains  98  grams  H.2S04  per  litre. 

2  This  acidity  is  equivalent  to  0'5  gram  of  H.2S04  per  litre. 


BESSON'S   METHOD  405 

After  filtration,  test  the  reaction  again  thus  :  mix  10  c.c.  of  gelatin  with  100  c.c. 
of  distilled  water,  add  a  few  drops  of  phenol-phthalein  solution,  and  from  a  1  c.c. 
pipette  graduated  in  tenths  of  a  cubic  centimetre  run  in  a  half-normal  solution  of 
soda  ;  the  red  colour  should  appear  when  0*2  c.c.  of  the  solution  have  been  added. 

The  desired  degree  of  acidity  being  obtained,  dissolve  2 '5  grams  of  magnesium 
sulphate  for  every  litre  of  gelatin.  Tube  in  quantities  of  10  c.c.  and  sterilize  on 
three  successive  occasions  at  100°  C. 

Immediately  before  use  add  to  each  tube  1  c.c.  of  a  sterile  35  per  cent,  solution  of 
lactose  and  O'l  c.c.  of  a  2'5  per  cent,  solution  of  carbolic  acid. 

Method  of  sowing. — Remy's  gelatin  is  used  in  the  same  way  as  Eisner's.  It  is 
advisable,  first  of  all,  to  sow  the  suspected  water  in  a  broth  containing  0*5  per  cent, 
of  sulphuric  and  carbolic  acids,  and  after  incubating  at  30°  C.  for  24  hours  to  use 
this  culture  for  sowing  the  gelatin  plates  by  the  dilution  method.  Colonies  of  the 
colon  and  typhoid  bacilli  appear  in  the  plates  after  incubating  for  2  days. 

Cultural  characteristics.  The  colon  'bacillus. — Colonies  in  the  depth  of  the  medium 
are  rounded,  ovoid  or  fusiform  and  of  a  yellowish- brown  colour.  Minute  bubbles 
of  gas  are  occasionally  formed.  Colonies  on  the  surface  which  are  sometimes  trans- 
parent and  bluish  at  first,  rapidly  become  opaque  :  some  of  them  are  hemispherical 
and  of  a  yellowish- brown  colour  while  others  have  irregular  margins  and  tend  to 
spread. 

The  typhoid,  bacillus. — In  the  depth  of  the  medium  the  colonies  are  bluish- white, 
smaller  than  the  colonies  of  the  colon  bacillus  and  form  no  gas.  Surface  colonies 
are  not  well  seen  until  the  third  day  :  at  first  "  they  are  rather  like  moulds  in  appear- 
ance "  but  later  spread  out,  become  more  bluish  in  colour  and  may  attain  the  size 
of  a  threepenny-piece. 

The  differences  between  the  colonies  of  the  typhoid  and  colon  bacilli  are  frequently 
very  slight  and  many  sub-cultures  may  have  to  be  made  before  the  nature  of  the 
organism  can  be  definitely  determined. 

D.  Besson's  method. 

Eisner's  gelatin  method  has  two  great  disadvantages.  In  the  first  place, 
it  is  only  available  for  the  analysis  of  small  quantities  of  water  even  though 
the  number  of  plates  used  be  large— which  is  in  itself  a  disadvantage — and, 
secondly,  the  medium  does  not  prevent  the  growth  of  saprophytic  organisms, 
which  sometimes  liquefy  the  plates  as  early  as  the  second  day  and  so  put  an 
end  to  the  experiment.  With  the  object  of  simplifying  and  at  the  same  time 
rendering  the  method  more  efficient  as  a  means  of  water  analysis,  Besson, 
in  1896,  introduced  certain  modifications  which  he  claims  improve  it  in  that 
they  rapidly  eliminate  saprophytes  and  permit  the  use  of  large  volumes 
of  water. 

1.  Weigh  out  30  grams  of  peptone  (Chapoteaut)  and  5  grams  common  salt, 
add  a  litre  of  water  and  dissolve  in  the  steamer ;  then,  without  neutralizing, 
heat  in  the  autoclave  to  115°  C.,  filter,  tube  in  quantities  of  10  c.c.  and 
sterilize  at  115°  C. 

2.  When  an  experiment  is  to  be  done,  take  ten  tubes  of  the  peptone  water 
and  to  each  add  20-30  drops  of  freshly  prepared  Gram's  iodine  solution 
(p.  143)  and  10  c.c.  of  the  water  to  be  examined. 

The  amount  of  iodine  solution  to  be  added  varies  a  little  with  the  composition  of 
the  peptone.  The  first  few  drops  will  be  rapidly  decolourized  but  when  about 
20-25  drops  have  been  added  the  medium  assumes  a  pale  brownish-pink  colour 
which  disappears  in  5-6  minutes.  When  this  occurs  sufficient  iodine  has  been 
added. 

3.  Incubate  the   tubes   at  37°-38°  C.     Under  these  conditions  the  colon 
bacillus  produces  a  visible  growth  in  8-12  hours  and  the  typhoid  bacillus 
in  about  15-20  hours  while  other  organisms  do  not  appear  until  later.     The 
tubes  should  be  examined  at  frequent  intervals. 

4.  After  incubating  for  18  hours  pick  out  the  tubes  which  are  cloudy  and 
sow  sub-cultures  in  iodine-peptone-water. 


406  ISOLATION  OF  THE  TYPHOID  BACILLUS 

5.  Incubate  the  latter  for  15  or  20  hours  then  plate  a  few  drops  from  each 
tube  on  litmus-lactose-agar.     At  the  same  time  sow  sub-cultures  in  ordinary 
broth  for  inoculation  later. 

6.  The  plates  are  to  be  examined  and  the  colonies  tested  as  described 
above. 

With  this  method  Besson  has  succeeded  in  isolating  the  colon  bacillus, 
the  typhoid  bacillus,  and  Friedlander's  bacillus  from  water. 

3.  Methods  based  on  precipitation. 

When  a  chemical  precipitate  is  produced  in  a  liquid  containing  micro-organisms, 
a  large  proportion  of  the  latter  are  carried  down  mechanically  with  the  precipitate, 
so  that  if  the  latter  be  collected  the  organisms  originally  present  in  the  liquid  are 
concentrated  in  a  small  volume.  The  principle  here  involved  is  the  basis  of  several 
methods  for  the  detection  of  the  colon  and  typhoid  bacilli  in  water.  Their  only 
advantage  is  in  the  concentration  of  the  micro-organisms,  since  the  nature  of  the 
organisms  and  the  presence  of  the  typhoid  bacillus  can  only  be  definitely  established 
by  carrying  out  a  series  of  experiments  on  ordinary  lines  using  the  precipitate  as  the 
original  material. 

A.  Vallet's  method. — Pour  20  c.c.  of  the  water  under  examination  into  a  sterile 
tube,  add  4  drops  of  a  saturated  aqueous  solution  of  hyposulphite  of  sodium  and  4 
drops  of  a  saturated  solution  of  lead  nitrate.     A  precipitate  forms  which  carries 
down  with  it  the  majority  of  the  organisms  present  in  the  water  (the  chemicals  used 
have  no  bactericidal  action  on  the  typhoid  bacillus).     Centrifuge  the  mixture  and 
suspend  the  deposit  in  a  few  drops  of  the  hyposulphite  solution.     Sow  the  liquid — 
which  now  contains  all  the  organisms  originally  present  in  the  20  c.c.  of  water — on 
Eisner's  gelatin  (vide  ante). 

B.  Schueder's  method. — In  Schueder's  method  the  fluid  is  not  centrifuged.     Pour 
2  litres  of  the  water  under  examination  into  a  tall  vessel  and  add  20  c.c.  of  a  7*75  per 
cent,  solution  of  hyposulphite  of  soda  and  20  c.c.  of  a  10  per  cent,  solution  of  lead 
nitrate.     Mix  intimately  and  allow  to  stand  for  20  hours.     Decant  the  supernatant 
liquid  and  suspend  the  precipitate  in  14  c.c.  of  a  saturated  solution  of  hyposulphite. 
Sow  the  emulsion  in  quantities  of  0'5  c.c.  on  a  number  of  litmus-lactose-agar 
plates. 

C.  Picker's  method. — Ficker  precipitates  the  organisms  with  sulphate  of  iron. 
To  2  litres  of  the  suspected  water  add  8  c.c.  of  a  10  per  cent,  solution  of  soda  and 
7  c.c.  of  a  10  per  cent,  solution  of  sulphate  of  iron.     The  precipitation  takes  2  or  3 
hours  to  complete.     Centrifuge  the  precipitate  and  dissolve  the  deposit  in  one- half 
its  volume  of  a  25  per  cent,  solution  of  neutral  tartrate  of  potassium.     Sow  the 
solution  on  Conradi  and  Drigalski's  medium  by  the  dilution  method  (p.  407). 

D.  Miiller's  method. — The  precipitation  is  effected  by  means  of  oxychloride  of 
iron  which  acts  more  quickly  than  the  sulphate  and  does  not  require  that  the  water 
shall  be  made  alkaline. 

To  3  Litres  of  the  water  under  examination  add  5  c.c.  of  the  oxychloride  solution. 
Precipitation  is  complete  in  about  half  an  hour.  Collect  the  precipitate  on  a  filter 
and  sow,  without  re-dissolving,  either  on  Conradi-Drigalski  plates  or,  better  (Meter), 
on  malachite-green-agar  plates  (p.  409). 

4.  Method  based  on  the  motility  of  the  typhoid  bacillus. 

Gambler,  in  examining  water  for  the  presence  of  typhoid  bacillus,  relies  upon  the 
property  possessed  by  the  organism  of  rapidly  passing  through  porous  membranes 
(p.  155). 

Place  a  porous  porcelain  bougie  in  a  large  test-tube.  Half  fill  both  the  bougie 
and  the  tube  with  ordinary  broth,  and  sterilize  in  the  autoclave.  Sow  the  suspected 
water  in  the  lumen  of  the  bougie  and  incubate  at  37°  C.  As  soon  as  the  broth  in  the 
test-tube  becomes  cloudy  sow  a  little  of  it  on  any  of  the  ordinary  media  used  for 
differentiating  the  typhoid  bacillus. 

This  method  is  not  very  reliable  since  some  strains  of  the  colon  bacillus  also  pass 


CONRADI  AND  DBIGALSKI'S  METHOD  407 

very  rapidly  through  the  walls  of  a  porous  bougie,  and  the  various  modifications  of 
the  method  which  have  been  introduced  seem  to  have  little  to  recommend  them. 

5.  Chantemesse's  methods. 

Chantemesse  has  introduced  two  methods  of  isolating  the  typhoid  bacillus, 
both  of  which  depend  upon  obtaining  surface  colonies  on  carbolic  agar.  For 
isolating  the  organism  from  stools  and  water  the  second  is  not  only  more 
rapid  but  is  simpler. 

First  method. — Filter  5  or  6  litres  of  the  suspected  water  through  a  Chamberland 
bougie.  Wash  the  surface  of  the  filter  in  200  c.c.  of  a  3  per  cent,  peptone  solution. 
Incubate  the  latter  at  37°  C.  and  arrange  the  culture  so  that  air  can  be  bubbled  through 
it  while  incubating.  Add  more  peptone  solution  at  the  end  of  12  hours  and  again 
at  the  end  of  24  hours.  Then  centrifuge  the  culture.  The  typhoid  bacilli  being 
motile  and  isolated  (i.e.  not  grouped  in  clumps)  remain  in  suspension  while  non- 
motile  organisms  and  those  massed  together  in  zooglea  masses  go  to  the  bottom  of 
the  vessel.  With  the  supernatant  liquid  sow  a  number  of  Esmarch's  roll  tubes  by 
the  dilution  method  using  carbolic-agar  as  the  medium. 

Carbolic-agar. — Dissolve  30  grams  of  peptone  and  20  grams  of  agar  in  a  litre  of 
water,  and  make  feebly  alkaline  (p.  31),  tube  in  quantities  of  10  c.c.  and  sterilize. 
Immediately  before  use  melt  the  agar  and  add  four  drops  of  a  5  per  cent,  solution  of 
carbolic  acid  to  each  tube  (  =0'1  per  cent,  of  carbolic  acid). 

Incubate  the  cultures  at  37°  C.  Growth  appears  in  about  16-20  hours.  Sow 
all  colonies  at  all  resembling  the  typhoid  bacillus  on  the  various  media  used  for 
differentiating  the  organism. 

Second  method  (recommended). — Sow  the  suspected  material  directly  on 
litmus-lactose-carbolic-agar. 

Litmus-lactose-carbolic-agar. — Prepare  agar  as  above  and  add  2  per  cent,  lactose. 
Tube  in  quantities  of  10  c.c.  and  sterilize.  Just  before  use  melt  a  number  of  tubes 
of  lactose-agar  and  to  each  add  1  c.c.  of  sterile  neutral  litmus  solution  and  4  drops 
of  a  5  per  cent,  solution  of  carbolic  acid.  Mix  thoroughly,  pour  into  Petri  dishes 
in  thin  layers  (1-2  mm.  deep)  and  allow  to  set. 

Dip  a  fine  sterile  badger-hair  brush  in  a  tube  of  sterile  water  to  which  a 
trace  of  the  suspected  stool  has  been  added,  and  without  recharging  it  sow 
in  succession  six  surface  plates  of  litmus-lactose-carbolic-agar. 

If  water  is  to  be  examined,  filter  it  through  a  Chamberland  bougie  and  sow 
litmus-lactose-carbolic-agar  plates  with  the  deposit  left  on  the  bougie.  For 
spreading  the  plates  use  a  glass  rod  bent  at  a  right  angle  (Drigalski's  spatula). 

Incubate  the  plates  at  37°  C.  and  after  12-15  hours  numerous  colonies  will 
be  found  on  the  plates  some  red  (the  colon  bacillus)  and  some  blue  (the  typhoid 
bacillus).  Test  the  blue  colonies  by  the  agglutination  reaction. 

6.  Conradi-Drigalski's  method. 

This  method,  in  principle  the  same  as  that  of  Chantemesse,  is  in  very  general 
use  in  Germany. 

The  suspected  material  is  sown  on  the  surface  of  agar  containing  lactose 
and  litmus.  Crystal- violet  is  used  in  place  of  the  carbolic  acid  in  Chante- 
messe's medium,  and  is  found  to  be  just  as  effective  in  restraining  the  majority 
of  organisms  while  allowing  the  growth  of  the  colon  and  typhoid  bacilli. 

Conradi  and  Drigalski's  medium. 

(a)  Preparation. — Macerate  1500  grams  of  minced  beef  in  2  litres  of  water  for 
24  hours  :  boil  the  mixture  for  an  hour :  filter :  make  up  to  2  litres  with  water : 
add— 

Peptone  (Witte),        ....  20  grams. 

Nutrose, 20 

Salt,         -  10 

boil,  add  60  grams  of  agar ;    heat  until  the  agar  is  dissolved  :    make  feebly  alkaline 


408  ISOLATION   OF   THE   TYPHOID   BACILLUS 

to  litmus  paper :   autoclave  for  an  hour  at  120°  C.,  filter  in  the  steamer  and  sterilize 
for  a  quarter  of  an  hour  at  115°  C.     Prepare 

Litmus  solution  (Kahlbaum),      -  -         300  c.c. 

Lactose,  -          -          -  30  grams. 

Sterilize  at  100°  C.  for  15  minutes. 

Mix  the  agar  and  litmus  solution  while  they  are  both  hot.  If  the  colour  of  the 
litmus  indicate  that  the  medium  is  acid  add  sufficient  10  per  cent,  soda  solution 
to  render  it  faintly  alkaline  and  then  a  further  4  c.c.  of  warm  10  per  cent,  solution 
of  sodium  hydroxide.  Lastly,  add  to  the  mixture,  20  c.c.  of  a  hot  sterile  (O'l  per 
cent.)  solution  of  crystal-violet  B,  Hochst. 

(/3)  Mode  of  use. — Pour  the  Conradi-Drigalski  agar  carefully,  without  contaminat- 
ing it,  into  large  Petri  dishes  (1,5-20  cm.  in  diameter).  Sow  the  suspected  material 
on  the  surface  of  the  agar  (vide  ante  Chantemesse's  method).  Incubate  the  plates 
at  37°  C.  The  typhoid  bacillus  gives  blue  transparent  colonies  and  the  colon  bacillus 
red  opaque  colonies. 

Hagemann's  medium. 
This  is  a  modification  of  the  preceding. 

Liebig's  extract  (Lemco),  -  10  grams. 

Peptone  (Witte),        -          -  10 

Salt,        -  3 

Water,     -  600  c.c. 

Boil.  Add  500  c.c.  of  milk.  Boil  and  dissolve  20  grams  of  agar  in  the  hot  liquid. 
Heat  to  120°  C.  in  the  autoclave  for  half  an  hour.  Filter  in  the  steamer  and  dis- 
tribute in  Erlenmeyer  flasks.  Sterilize.  When  required  for  use,  liquefy  the  agar, 
make  slightly  alkaline  with  soda  solution,  add  a  few  cubic  centimetres  of  litmus 
and  finally  three  drops  of  a  1  per  cent,  alcoholic  solution  of  crystal  violet. 

7.  Method  of  Endo. 

The  principle  of  the  method  depends  upon  the  fact  that  if  sulphite  of  sodium 
be  added  to  agar  containing  fuchsin  the  medium  is  decolourized,  and  if  the 
decolourized  medium  be  sown  with  the  colon  bacillus  the  acids  produced  by 
the  organism  restore  the  colour  of  the  fuchsin  and  the  colonies  of  the  organism 
acquire  a  red  colour,  while  under  similar  conditions  the  colonies  of  the  typhoid 
and  paratyphoid  bacilli  are  colourless. 

The  agar  medium  of  Endo  is  used  in  exactly  the  same  way  as  Chantemesse's 
and  Conradi-Drigalski's  agar.  It  is  very  easy  to  prepare  and  gives  good 
results. 

Prepare  a  litre  of  peptone  broth  in  the  ordinary  way,  add  30  grams  of  agar  and 
dissolve  in  the  steamer.  Filter.  Make  absolutely  neutral  to  litmus  paper  then  add 
10  c.c.  of  a  10  per  cent,  solution  of  sodium  bicarbonate.  * 

Add  10  grams  of  chemically  pure  lactose,  and  5  c.c.  of  a  filtered  saturated  alcoholic 
solution  of  fuchsin  which  imparts  a  red  colour  to  the  medium.  Now  add  25  c.c.  of 
a  freshly  prepared  10  per  cent,  solution  of  sodium  sulphite.  Decolourization  com- 
mences at  once  and  is  complete  after  sterilization.  Distribute  in  quantities  of  15  c.c. 
in  tubes,  sterilize  at  115°  C.  and  store  in  the  dark.  When  required  for  use  melt 
the  agar  and  pour  into  Petri  dishes. 

After  incubating  for  15  hours  at  37°  C.  colonies  of  the  colon  bacillus  on  this 
medium  have  red  centres  and  after  24  hours  are  entirely  red  with  a  greenish 
iridescence. 

8.  Methods  based  on  the  use  of  caffeine. 

As  already  mentioned  (p.  375)  Roth  has  shown  that  the  addition  of  0'5  per  cent, 
of  caffeine  to  culture  media  checks  the  growth  of  the  colon  bacillus  but  does  not 
interfere  with  that  of  the  typhoid  bacillus.  This  fact  has  been  successfully  applied 
by  Roth,  Hoffmann  and  others  to  the  isolation  of  the  typhoid  bacillus  from  water 
and  stools.  According  to  Courmont  and  Lacomme  however,  the  method  is  uncer- 
tain since  some  strains  of  the  typhoid  bacillus  do  not  grow  on  caffeine- containing 
media.  The  results  should  always  be  controlled  by  some  other  method. 


MALACHITE   GREEN   MEDIA  409 

Roth's  technique. — Prepare  broth  in  the  ordinary  way  and  add  sufficient  soda 
solution  to  give  a  permanent  pink  colour  with  phenol- phthalein.  Add  80-100  c.c. 
of  a  1  per  cent,  solution  of  caffeine  to  every  100  c.c.  of  broth. 

Sow  the  fluid  with  the  material  to  be  examined  and  incubate  at  37°  C.  for  24  hours, 
then  plate  traces  of  the  culture  on  gelatin. 

Picker's  technique. — To  100  c.c.  of  a  3  per  cent,  peptone-meat-broth  add  0'6  gram 
of  pure  caffeine  and  0*00007  gram  of  crystal- violet  (0'7  c.c.  of  a  O'Ol  per  cent,  solution). 
Sow  the  fluid  with  the  suspected  material,  incubate  at  37°  C.  for  12  or  13  hours  and 
sow  Conradi-Drigalski  plates  with  the  culture  obtained. 

Lubenau's  technique. — Lubenau  sows  in  100  c.c.  of  Ficker's  broth  containing 
0-3  per  cent,  of  caffeine,  incubates  for  13  hours,  adds  100  c.c.  of  broth  containing  0*6 
per  cent,  of  caffeine,  incubates  for  a  second  period  of  13  hours  and  adds  100  c.c.  of 
broth  containing  0*9  per  cent,  of  caffeine.  Before  and  after  the  second  addition  of 
broth  he  sows  surface  plate  cultures  on  litmus-lactose-caffeine-agar  for  purposes 
of  isolation. 

Lubenau's  caffeine-cigar. — Prepare  a  litre  of  6  per  cent,  peptone- beef -broth, 
dissolve  40—60  grams  of  agar  in  the  broth,  make  neutral  to  litmus,  heat  to  120°  C., 
filter  and  sterilize.  After  sterilization  and  while  the  agar  is  still  hot  add  60  c.c.  of 
litmus  solution,  5  grams  of  lactose  and  finally  110  c.c.  of  a  6  per  cent,  solution  of 
pure  caffeine.  Distribute  in  Petri  dishes. 

Gathgens'  technique. — To  a  litre  of  Endo's  medium  add  33  c.c.  of  a  10  per  cent, 
solution  of  pure  caffeine.  Distribute  in  tubes  hi  quantities  of  15  c.c.  which  can 
afterwards  be  used  to  prepare  plates, 

9.  Malachite  green  media. 

Loeffler  has  stated  that  the  addition  of  a  certain  quantity  of  malachite 
green  to  culture  media  impedes  the  development  of  the  colon  bacillus  while 
having  no  effect  on  the  growth  of  the  typhoid  and  paratyphoid  bacilli. 

Unfortunately  malachite  green  media  are  not  so  selective  as  Loeffler  believed, 
for  though  the  growth  of  a  large  number  of  organisms  (streptococci,  staphy- 
lococci,  cholera  vibrios,  etc.)  is  inhibited  the  colon  bacillus  will  often  grow 
(Kiralyfi). 

These  media  are  difficult  to  prepare  ;  if  too  much  green  be  added  the 
typhoid  bacillus  is  inhibited,  if  too  little,  the  colon  bacillus  grows  as  rapidly 
as  the  typhoid  bacillus.  It  is  essential  to  use  a  chemically  pure  compound 
and  the  amount  to  be  added  to  the  agar  varies  very  much  with  the  different 
commercial  preparations.  A  series  of  experiments  should  be  done  to  deter- 
mine the  quantity  (1  in  4,000  to  1  in  6,000)  to  be  added  (Lentz  and  Tietz, 
Schindler).  These  different  complications  render  the  method  of  little  practical 
value,  and  to  make  it  more  efficient  Loeffler  has  recently  advised  the  addition 
of  ox  bile  to  his  malachite  green  media  (vide  infra).  As  the  result  of  his  own 
experience  Fiirth  concludes  that  methods  based  upon  the  use  of  malachite 
green  are  inferior  to  Conradi-Drigalski's  method. 

Growth  on  malachite  green  media  diminishes  the  agglutinability  of  the 
typhoid  bacillus. 

Of  the  different  malachite  green  methods  Leuch's  seems  to  be  the  best. 

Leuch's  technique. — Prepare  an  agar  medium  with  : — 

Beef,        ...  -         500  grams. 

Water,     -  1  litre. 

Common  salt,  -  5  grams. 

Dextrin.  -  10         „ 

Agar,       -  -         -     30-40 

Neutralize,  using  litmus  as  the  indicator.  Add  5  c.c.  of  a  normal  solution  of 
sodium  carbonate  and  100  c.c.  of  a  10  per  cent,  solution  of  nutrose.  After  filtering 
and  sterilizing  add  16-18  c.c.  of  a  1  per  cent,  solution  of  malachite  green. 

Surface  cultures  are  sown  in  Petri  dishes  (p.  407).  Colonies  of  the  typhoid  bacillus 
destroy  the  colour  of  the  medium  and  a  characteristic  yellowish  zone  forms  around 
them. 


410  ISOLATION   OF   THE   TYPHOID   BACILLUS 

To  identify  an  organism  isolated  on  this  medium  Loeffler  advises  the  use  of  a 
so-called  typhoid  solution. 

Loeffler" s  typhoid  solution. — This  fluid  is  coagulated  by  the  typhoid  bacillus  in 
16-24  hours  and  floating  on  the  coagulum  is  a  clear  green  liquid.     The  colon  bacillus 
produces  not  an  homogeneous  coagulum  but  a  greenish  mass  adhering  to  the  sides 
of  the  tube. 

The  solution  consists  of  : 

Distilled  water,  100     c.c. 

Nutrose,  gram. 

Glucose,  -  » 

Peptone,  2     grams. 

Lactose,  ------  5          ., 

2  per  cent,  solution  of  chemically  pure  malachite  green,          -  1     c.c. 

Normal  soda  solution,        -  1'5    ., 

Peabody  and  Pratt's  technique. — Sow  the  suspected  fluid  in  broth  con- 
taining O'l  per  cent,  of  malachite  green,  incubate  at  37°  C.  for  18  hours  and 
isolate  on  Conradi-Drigalski  plates. 

10.  Method  based  upon  the  use  of  China  green. 

[Werbitzki  recommends  the  addition  of  China  green  to  agar  (I'l-l'S  c.c. 
of  a  0*2  per  cent,  solution  per  100  c.c.  of  agar)  for  the  purpose  of  restraining 
the  growth  of  the  colon  bacillus  when  attempting  the  isolation  of  the  typhoid 
and  paratyphoid  bacilli  from  such  material  as  stools.] 

11.  Methods  based  on  the  use  of  bile. 

The  adjuvant  properties  of  bile  for  the  typhoid  bacillus  have  been  applied 
to  the  detection  of  the  bacillus  in  water  and  stools.     Bile  may  be  used  alone 
(either  as  such  or  in  the  form  of  bile  salts)  or  mixed  with  malachite  green. 
Diinschmann's  technique. — Prepare  the  following  medium : 

Distilled  water,  -         100  c.c. 

Agar,       -  3  grams. 

Peptone,  3         „ 

Lactose,  -  3         , 

Gelatin,  -  1  gram. 

Taurocholate  of  sodium,    -  1        „ 

Heat  to  120°  C.  Filter  and  distribute  the  medium  in  tubes  in  quantities  of  10  c.c. 
After  sterilization,  add  1  c.c.  of  a  sterile  solution  of  litmus  to  each  tube. 

To  use  the  medium,  pour  the  contents  of  four  or  five  tubes  into  a  similar  number 
of  Petri  dishes  and  with  a  Drigalski's  spatula  (p.  407)  charged  with  the  suspected 
material — and  without  recharging — sow  surface  cultures  on  each  dish. 

Jackson  and  Melia's  technique. — The  suspected  material  is  enriched  by  growing 
in  ox  bile  and  the  culture  used  to  sow  plates  of  Hesse's  agar. 
Hesse's  agar. — In  a  litre  of  boiling  water  dissolve  : 

Liebig's  extract,        -  5  grams. 

Peptone,  10         „ 

Sodium  chloride,        -  8'5      ,, 

Agar,       -  30 

Heat  to  120°  C.,  filter,  distribute  in  tubes  (10  c.c.  in  each)  and  sterilize  in  the 
autoclave. 

Method. — Sow  the  suspected  material  in  5  c.c.  of  bile  and  incubate  at  37°  C.  for 
24  hours.  Take  eight  test-tubes  each  containing  9  c.c.  of  sterile  water :  to  the  first 
add  1  c.c.  of  the  bile  culture,  to  the  second  1  c.c.  of  the  first,  to  the  third  1  c.c.  of 
the  second  and  so  on.  Take  eight  tubes  of  Hesse's  agar,  liquefy  the  medium  and  to 
one  add  1  c.c.  of  the  first  dilution  to  the  second  1  c.c.  of  the  second  dilution  and  so 
on,  and  pour  plates. 

Lceffler's  technique. — A  mixture  of  bile  and  malachite  green  is  used.  Sow  the 
material  on  plates  of  Leuchs'  nutrose-agar  (p.  409)  containing  3  per  cent,  of  ox  bile 
and  1-9  per  cent,  of  a  0*2  per  cent,  solution  of  malachite  green. 


NEUTRAL   RED   MEDIA  411 

Padlewsky's  technique. — This   method  also  depends  upon  the  use  of  a  mixture 
of  bile  and  malachite  green.     Sow  on  plates  prepared  with  the  following  medium  : 


Distilled  water, 
Agar,       - 
Peptone, 
Ox  bile,  - 
Lactose,  - 


100  c.c. 
3  grams. 
3 
5 

1  gram. 


The  medium  should  be  slightly  alkaline  to  litmus.  After  sterilization  cool  to 
65°  C.  and  add,  firstly  a  mixture  of 

1  per  cent,  aqueous  solution  of  malachite  green,  -  0'5  c.c. 

Ox  bile,  -  0-5      „ 

then, 

10  per  cent,  aqueous  solution  of  sulphite  of  sodium.  -  1  c.c. 

12.  Method  based  upon  the  use  of  brilliant  green. 

Conradi  substitutes  for  malachite  green  a  mixture  of  Brillantgrun-Kristall 

and  picric  acid.     An  agar  containing  these  dyes  favours  the  growth  of  the 

typhoid  and  paratyphoid  bacilli  while  inhibiting  that  of  most  other  organisms. 

The  colon  bacillus  either  does  not  grow  at  all  or  only  in  very  small  numbers. 

Prepare  a  slightly  modified  Hesse's  agar : 

Water,  1  litre. 

Peptone,  -  10  grams. 

Agar,       -  30        „ 

Liebig's  extract,         -  20 

Make  alkaline,  heat  and  filter.     For  every  1*5  litres  of  agar  add 
O'l  per  cent,  aqueous  solution  of  Brillantgrun-Kristall  (extra 

pure,  Hochst),    -  10  c.c. 

1  per  cent,  aqueous  solution  of  picric  acid,  -  10     ,, 

On  this  medium,  the  colonies  of  the  typhoid  bacillus  are  bright  green  and  trans- 
parent and  thicker  in  the  centre  than  at  the  margins  :  colonies  of  the  paratyphoid 
bacilli  are  larger,  and  yellowish-green  in  colour. 

13.  Method  based  upon  the  use  of  neutral  red. 

Savage  has  applied  the  property  possessed  by  the  colon  bacillus  of  reducing 
neutral  red  to  the  detection  of  that  organism  in  water  (p.  397). 

The  method  is  only  applicable  to  the  detection  of  the  colon  bacillus  and 
does  not  indicate  the  presence  of  the  typhoid  bacillus. 

The  reduction  of  neutral  red,  however,  is  not,  as  was  formerly  thought  to 
be  the  case,  a  specific  property  of  the  colon  and  paratyphoid  bacilli :  E.  pyo- 
cyaneus,  B.  fluorescens,  B.  enteritidis  and  some  of  the  harmless  saprophytic 
organisms  found  in  water  give  fluorescence  in  Savage's  neutral-red  broth  ; 
while,  on  the  other  hand,  some  strains  of  the  colon  bacillus  exert  hardly  any 
decolourizing  action  on  neutral  red  (Sicre,  Vincent).  Savage's  method  is 
therefore  unreliable. 

Savage's  technique. — Prepare  broth  thus  : — 

Water, 1  litre. 

Beef,        -  -         250  grams. 

Boil,  make  up  to  1  litre  and  add 

Peptone  (Defresne),  -  20  grams. 

Common  salt,  -  20         „ 

Glucose,  -  5         „ 

Boil,  cool,  decant  and  add  10  c.c.  of  a  5  per  cent,  solution  of  neutral-red.  Dis- 
tribute in  tubes  and  sterilize.  The  medium  should  be  ruby-red  in  colour. 

Method  of  analysis. — Sow  a  number  of  tubes  of  the  medium  with  different  quan- 
tities (1  c.c.  to  10  c.c.)  of  the  suspected  water.  Incubate  at  37°  C.  for  24  hours. 
The  presence  of  the  colon  bacillus  is  indicated  by  a  beautiful  green  fluorescence  or  a 
canary  yellow  tint  according  as  to  whether  the  water  contains  few  or  many  colon  bacilli. 


412  ISOLATION   OF  THE   TYPHOID  BACILLUS 

14.  Methods  based  on  the  agglutination  of  the  typhoid 

bacillus. 

Chantemesse,  Windelbandt,  Schepilewsky  have  made  use  of  the  agglutinat- 
ing properties  of  antityphoid  serum  for  isolating  the  typhoid  bacillus.  This 
method  is  very  delicate  and  permits  of  the  isolation  of  the  bacillus  from 
mixtures  in  which  it  is  present  in  great  dilution.  - 

Windelbandt' s  technique. — To  10  c.c.  of  sterile  broth  add  1  c.c.  of  the  water  under 
examination.  Incubate  the  mixture  for  3—5  days.  By  this  time  the  growth  is  very 
abundant,  the  broth  is  cloudy  and  the  surface  covered  with  a  pellicle.  Remove  the 
surface  growth  and  add  to  the  remainder  a  few  drops  of  a  powerfully  agglutinating 
anti-typhoid  serum.  The  agglutinated  typhoid  bacilli  fall  to  the  bottom  of  the 
tube.  Centrifuge  the  broth  culture,  collect  the  deposit  and  dilute  it  with  a  little 
normal  saline  solution.  Sow  litmus-lactose-agar  plates  with  the  diluted  deposit. 

Chantemesse' s  technique. — A  simple  method  devised  by  Chantemesse  consists  in 
adding  30  grams  of  peptone  to  1  litre  of  the  suspected  water,  neutralizing  and 
incubating  for  20  hours.  If  little  clumps  form  filter  through  paper  and  add  anti- 
typhoid serum  to  the  filtrate.  After  standing  for  2  hours  decant  the  liquid,  filter 
the  deposit  through  paper,  and  sow  the  clumps  retained  on  the  filter  on  Chantemesse' s 
agar  (p.  407). 

Altschiiller's  technique. — Altschliller  adds  peptone  and  salt 'to  the  suspected  water 
and  incubates  for  24  hours.  Ten  c.c.  of  the  culture  are  now  transferred  to  a  test- 
tube  the  lower  end  of  which  is  drawn  out  and  opened  and  attached  to  a  piece  of 
india-rubber  tubing  closed  by  a  clip.  A  few  drops  of  a  typhoid  immune  serum  are 
added  and  a  precipitate  is  soon  formed  which  collects  in  the  narrow  drawn-out  part 
of  the  tube.  By  releasing  the  clip  the  deposit  can  be  run  into  a  tube  containing 
sterile  peptone  water.  The  mixture  is  shaken  and  then  incubated  at  37°  C.  The 
typhoid  bacillus  grows  rapidly  and  is  unaccompanied  by  other  organisms. 

[15.  MacConkey's  media.] 

[The  basis  of  MacConkey's  media  consists  of  a  stock  solution  composed  of  : 
Sodium  taurocholate  (commercial),      -  0'5  gram. 

Peptone  (Witte),       -         -  2'0  grams. 

Distilled  water,  -         100          „ 

For  liquid  media  there  is  added  to  this  stock  solution  0'5  per  cent,  of  a  1  per  cent, 
solution  of  neutral  red  and  0'5  per  cent,  of  glucose  or  1  per  cent,  of  the  other  carbo- 
hydrates or  alcohols,  as  the  case  may  be,  and  the  medium  is  distributed  into  Durham's 
fermentation  tubes  and  sterilized  in  the  steamer  for  10  minutes  on  each  of  two  days, 
great  care  being  taken  not  to  overheat  the  medium.  If  it  be  thought  advisable 
white  of  egg  may  be  used  to  clear  the  medium. 

[Bile-salt-agar  is  made  by  dissolving  1*5-2  per  cent,  agar  in  the  stock  solution. 
This  is  best  done  in  the  autoclave.  The  medium  is  cleared  with  white  of  egg  and 
filtered.  After  filtration  the.  same  amount  of  neutral  red  is  added  as  in  the  case  of 
the  liquid  media  (MacConkey). 

[A  consideration  of  the  fermentation  reactions  of  the  various  organisms 
shows  that  by  the  use  of  certain  carbohydrates  or  alcohols  either  alone  or  in 
combination  organisms  can  be  separated  by  means  of  colour  reactions.  Mac- 
Conkey's medium  forms  a  most  useful  nutritive  medium  to  which  to  add 
these  substances.] 


SECTION  II.— THE   IDENTIFICATION  OF  THE  TYPHOID   AND 
COLON   BACILLI. 

(i)  An  organism  may  be  suspected  to  belong  to  the  typhoid-colon  group 
if  it  have  the  following  characteristics  : 

1.  A  bacillus  with  rounded  ends,  generally  motile,  decolourized  by  Gram's 
method,  having  no  capsule  [and  not  forming  spores]. 


IDENTIFICATION   OF  THE  TYPHOID  BACILLUS        413 

2.  Cloudiness  with,  a  watered-silk  appearance  in  broth  culture. 

S.  No  liquefaction  of  gelatin. 

(ii)  If  conforming  to  these  requirements  it  remains  to  determine  if 
the  organism  (which  must  of  course  be  investigated  in  pure  -culture)  be  a 
typhoid  or  colon  bacillus. 

It  is  now  that  difficulties  arise,  though  if  what  has  been  said  in  the  fore- 
going chapters  be  recalled  it  seems  impossible  to  confuse  typical  specimens 
of  the  two  organisms.  The  motility,  the  characters  of  the  flagella,  the 
appearance  of  the  growth  on  potato,  the  indol  reaction,  a  study  of  the  fer- 
mentation properties  and  the  agglutination  test  should  furnish  a  sure  means 
of  diagnosis. 

Unfortunately  some  strains  of  the  colon  bacillus  readily  lose — when  grown, 
for  instance,  symbiotically  with  the  typhoid  bacillus  (Remy)  or  a  Pasteurella 
(Lesage),  etc. — their  capacity  to  produce  indol  and  some  of  their  fermenta- 
tion properties  ;  other  strains  are  very  motile,  while  others  again  yield  a 
very  scanty  growth  on  potato  and  on  gelatin  grow  like  the  typhoid  bacillus. 
Similarly,  some  strains  of  the  typhoid  bacillus  are  only  slightly  motile,  and 
their  flagella  can  only  be  stained  with  difficulty  :  others  give  a  slightly 
pigmented  growth  on  potato  resembling  cultures  of  the  colon  bacillus  : 
finally,  when  grown  in  the  presence  of  the  colon  bacillus  or  after  passing 
through  human  tissues  some  strains  lose  their  characteristic  property  of 
being  agglutinated  by  antityphoid  serum.  Hence  a  certain  amount  of  con- 
fusion arises  which  is  further  increased  by  the  existence  of  a  whole  group 
of  bacilli  very  closely  related  to  the  typhoid  bacillus  and  having  properties 
intermediate  between  it  and  the  colon  bacilli  (paratyphoid  bacilli,  Chap. XXV.). 

It  will  therefore  be  clear  that  for  accurate  diagnosis  it  is  necessary  to  study 
several  of  the  characteristics  of  the  organism.  The  table  below  gives  a  list 
of  the  tests  on  which  the  diagnosis  should  be  based.  The  investigation  of 
the  fermentation  reactions,  the  production  of  indol,  the  characters  of  the 
flagella  and  the  agglutinating  properties  will  in  the  majority  of  cases  afford 
sufficient  information  upon  which  to  determine  whether  the  organism  is  a 
typhoid  or  a  colon  bacillus.  When  an  organism  has  all  the  characteristics 
of  the  typhoid  bacillus  except  that  it  is  not  agglutinated  by  an  antityphoid 
serum  it  must  be  tested  as  indicated  under  14  in  the  table.  If  the  serum  of 
a  guinea-pig  which  has  been  inoculated  every  other  day  for  a  fortnight  with 
2  c.c.  of  a  forty-eight-hour  old  broth  culture  of  the  organism  under  investiga- 
tion agglutinate  an  authentic  typhoid  bacillus  in  a  dilution  of  at  least  1  in 
40  (Remy)  the  organism  must  be  regarded  as  a  strain  of  the  typhoid  bacillus. 
Finally  the  two  last  tests  (15  and  16)  in  the  table  will  be  found  very  valuable 
and  should  permit  of  the  identification  of  the  organism  in  even  the  most 
difficult  cases. 


METHOD  OP  DIAGNOSIS. 


COLON  BACILLUS. 


TYPHOID  BACILLUS. 


1.  Culture  in  carbonated 
lactose-broth. 

2.  Stroke  culture  on  lit- 
mus-lactose-gelatin. 


3.  Single  colonies  on  lit- 
mus-lactose-agar. 


Abundant  gas -formation 
(12-36  hrs.). 

The  colour  of  the  litmus 
is  first  changed  to  red 
then  to  a  pale  brown 
along  the  stroke. 

Red  colonies. 


No  gas  formation. 

The  colour  of  the  litmus 
is  unchanged. 


Blue  or  violet  colonies. 


414        IDENTIFICATION   OF   THE   TYPHOID   BACILLUS 


METHOD  OF  DIAGNOSIS. 


COLON  BACILLUS. 


TYPHOID  BACILLUS. 


4.  Growth  in  milk. 


5.  Growth  on  potato. 


6.  Action  on  neutral-red. 

7.  Growth  on  artichoke. 


Growth  on  the  syn- 
thetic media  of  Ncegeli, 
Remy  and  Sugg, 
Frankel  and  others. 


9.  Growth 
water. 


in      peptone 


10.  Single  colonies  on  de- 
colourized  fuchsin-agar 
(Endo). 

11.  Growth  on  (a)  Caffe- 
ine media.     (6)  Malach- 
ite green. 

12.  Flagella. 


13.  Action  of  anti-typhoid 
serum  (using  the  serum 
in  its  highest  agglutinat- 
ing dilution). 

14.  Serum  of  a  guinea-pig 
immunized     with     the 
organism. 


15.  Simultaneous  inocula- 
tion of  anti-typhoid 
serum. 


16.  Complement  fixation. 


Coagulation    in     24 
hours.1 


36 


Thick  brownish  growth 
(inconstant). 

Reduced. 

Abundant  growth,  the 
medium  becoming 
green  (inconstant). 

Copious  and  rapid  growth 
(inconstant). 


Indol. 


Red  colonies. 


No  growth  (possible  ex- 
ceptions). 


Flagella  short  and  few 
in  number  (3  to  4  on 
each  bacillus). 

No  agglutination. 


The  serum  does  not  ag- 
glutinate a  true  typhoid 
bacillus  in  a  dilution  of 
1  in  40. 


If  the  bacillus  is  virulent 
the  simultaneous  inoc- 
ulation of  antityphoid 
serum  does  not  pro- 
tect the  animal. 

No  deviation  of  comple- 
ment with  a  heated 
antityphoid  serum. 


No  coagulation. 

Thin     colourless     glazed 
growth  (inconstant). 

Not  reduced. 

No    apparent    growth. 
No  change  in  colour  of 
medium. 

A   poor  growth   appear- 
ing slowly  (inconstant). 


No  indol. 


Colourless  colonies. 


Growth  (possible  excep- 
tions). 


Numerous  (8-18),  long, 
wavy,  undulating  fla- 
gella. 

Distinct    agglutination 
(possible  exceptions).2 


The  serum  agglutinates 
a  true  typhoid  bacillus 
in  a  dilution  of  1  in  40 
(some  possible  excep- 
tions). 

If  the  bacillus  is  viru- 
lent, the  simultaneous 
inoculation  of  anti- 
typhoid serum  pro- 
tects the  animal. 

Complement  is  deviated 
with  a  heated  anti- 
typhoid serum. 


1  Should  no  coagulation  occur  sow  the  bacillus  in  a  shallow  layer  of  milk.     Some 
strains  of  the  colon  bacillus  only  coagulate  milk  under  these  conditions. 

2  Typhoid  bacilli  recently  isolated  from  the  body  occasionally  fail  to  agglutinate  until 
they  have  been  sub-cultured  several  times  in  broth. 


CHAPTEE  XXIV. 
THE  PNEUMOBACILLUS  OF  FRIEDLANDEK. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  416. 

Section  II. — Morphology,  p.  416. 

Section  III. — Biological  properties,  p.  417. 

Section  IV. — Detection  and  isolation  of  the  pneumobacillus,  p.  418. 

The  bacillus  of  rhinoscleroma,  p.  418. 

The  bacillus  of  ozsena,  p.  419. 

THOUGH  the  pneumobacillus  is  not — as  Friedlander  believed — the  infect- 
ing agent  in  acute  lobar  pneumonia,  it  nevertheless  occupies  an  important 
place  in  human  pathology  and  may  be  the  cause  of  any  of  the  following  dis- 
eases, viz.  :  broncho-pneumonia,  pericarditis,  pleurisy,  peritonitis  meningitis, 
otitis,  parotiditis,  dacryocystitis,  stomatitis,  orchitis,  and  epididymitis  ;  and 
is  further  responsible  for  many  suppurative  conditions.  Ch.  Nicolle  and 
Hebert  have  drawn  attention  to  the  fact  that  some  cases  of  pseudo-mem- 
branous sore  throats  are  due  to  the  pneumobacillus  ;  it  is  also  associated  at 
times  with  the  diphtheria  bacillus  ;  and  finally,  it  may  occasionally  cause 
an  hsemorrhagic  type  of  septicaemia  (Weichselbaum,  Netter). 

It  is  present  in  the  saliva  of  many  persons  (4 '5  per  cent,  according  to 
Netter).  In  the  circumambient  media  the  bacillus  appears  to  be  widely 
distributed  ;  Uffelmann  found  it  in  the  air,  Emmerich  in  dust,  Grimbert  in 
water,  Besson  in  samples  of  water  from  many  and  various  sources. 

No  valid  distinction  can  now  be  drawn  between  the  pneumobacillus  and 
the  bacillus  described  by  Escherich  as  the  Bacillus  lactis  aerogenes  :  the 
proof  of  their  identity  was  sketched  by  Denys  and  Martin  and  extended  by 
Grimbert  and  Legros.1  These  researches  were  confirmed  by  Bertarelli ;  he 
considered  the  Bacillus  lactis  aerogenes  to  be  merely  a  variety  of  the  pneumo- 
bacillus. 

The  Bacillus  lactis  aerogenes  has  been  found  in  stools,  in  soil,  water,  and  air.  It 
is  one  of  the  causes  of  the  fermentation  of  milk  and  seems  to  be  responsible  for  some 
cases  of  enteritis  in  breast-fed  children.  It  plays  an  important  role  in-  urinary 
infections  (Morelle,  Worsburg,  Heyse,  etc.). 

1  Without  enlarging  upon  the  facts  which  have  led  to  the  conclusion  that  the  two 
organisms  are  identical,  the  following  characters  which  according  to  Grimbert  and  Legros 
they  possess  in  common  may  just  be  mentioned.  They  are  both  non-motile  encapsulated 
bacilli,  do  not  liquefy  gelatin,  do  not  produce  indol,  ferment  the  same  sugars  and  have 
the  same  action  upon  animals. 


416  THE  PNEUMOBACILLUS 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

Mice  and  guinea-pigs  are  very  susceptible  to  the  inoculation  of  virulent 
strains  of  the  pneumobacillus  (vide  infra).  Rabbits  are  distinctly  more 
immune. 

Mice. — If  a  few  drops  of  a  culture  be  inoculated  sub-cutaneously  into  a 
mouse  they  lead  to  the  formation  of  an  abscess  containing  creamy,  ropy  pus  ; 
the  bacillus  then  becomes  generalized  and  the  animal  dies  in  1-3  days.  Post 
mortem,  the  spleen  is  enlarged  and  the  bacillus  can  be  isolated  from  the  blood 
and  internal  organs.  Intra-pulmonary  inoculation  results  in  the  formation 
of  foci  of  broncho-pneumonia  and  terminates  in  death. 

Guinea-pigs. — Sub-cutaneous  inoculation  of  a  small  dose  of  culture  leads 
to  the  formation  of  an  abscess  at  the  site  of  inoculation.  Doses  of  1  c.c.  of 
a  broth  culture  prove  fatal :  an  abscess  forms  at  the  site  of  inoculation  and 
death  supervenes  more  or  less  rapidly  with  lesions  of  broncho-pneumonia 
and  generalization  of  the  bacillus. 

Rabbits. — A  dose  of  several  c.c.  of  a  broth  culture  injected  into  the  marginal 
vein  of  the  ear  of  a  rabbit  leads  to  the  death  of  the  animal  in  a  few  days. 
The  bacillus  may  be  found  in  the  blood  and  internal  organs  :  lesions  of 
hsemorrhagic  septicaemia  are  sometimes  present.  Sub-cutaneous  inoculation 
is  followed  by  a  less  severe  disease. 

Ch.  Nicolle  and  Hebert  by  abrading  the  mucous  membrane  of  the  vulva 
of  a  rabbit  and  infecting  the  abraded  area  produced  a  swelling  of  the  labia 
majora  which  was  accompanied  by  a  white  discharge  rich  in  pneumobacilli. 

Pigeons. — Pigeons  are  only  slightly  susceptible.  The  inoculation  of  very 
virulent  strains  into  the  peritoneum  is  however  fatal. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  pneumobacillus  is  a  rather  broad,  rod-shaped  organism  of  which  the 
length  does  not  exceed  on  an  average  1-2/x.     Sometimes  however  in  cultures, 
^  besides  the  cocco-bacillary  forms,  other  long  and 

even  filamentous  bacilli  may  be  seen.     The  bacilli 
\  are  often  arranged  in  pairs :  they  are  non-motile 

and  never  form  spores. 

In  pus,  sputum  and  blood,  the  pneumobacillus 
has  a  distinct  capsule.  The  capsule  is  less  dis- 
tinct, but  can  nevertheless  be  demonstrated,  in 
artificial  cultures  on  solid  media  (Grimbert, 
Nicolle  and  Hebert). 

Staining    reactions. — The    pneumobacillus    is 
easily  stained  by  the  basic  aniline  dyes.     It  is 
gram-negative.     The   capsules   may   be    stained 
Vti^thpd  described  in  the  chapter  dealing 
thionin.     x  iooo.)  with  the  pneumococcus. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  pneumobacillus  is  a  facultative  anaerobe  and 
grows  on  all  the  ordinary  media,  which  should  be  slightly  acid  for  preference. 
Cultures  can  be  obtained  above  15°  C.  ;  the  optimum  temperature  is  about 
37  C. 

Characters  of  growth  on  the  ordinary  media.    Broth.— After  incubating 


BIOLOGICAL  PROPERTIES  417 

for  24  hours  at  37°  C.  the  medium  is  slightly  cloudy,  and  on  the  surface  a 
viscous  pellicle  is  formed  which  makes  a  ring  round  the  tube  just  above  the 
surface  of  the  liquid.  On  further  incubation  the  pellicle 
falls  to  the  bottom  of  the  tube  leaving  the  broth  cloudy 
and  viscous. 

Gelatin.  Stab  culture. — Incubated  at  20°  C.,  a  small 
raised  white  growth  is  formed  on  the  surface  of  the  gelatin 
after  48  hours  :  the  growth  later  extends  along  the  line 
of  the  stab  and  assumes  a  typical  nail-line  appearance. 
The  medium  is  not  liquefied.  Bubbles  of  gas  often  form 
around  the  growth. 

Isolated  colonies. — Small  round  granular  whitish  colonies, 
which  become  somewhat  raised,  appear  towards  the  third 
day. 

Agar.  Coagulated  serum. — Growth  on  these  media  takes 
the  form  of  a  thick  white  viscous  layer. 

Potato. — A  thick  yellowish  and  viscous  streak  is  formed 
and  gas  is  also  produced. 

Milk. — The  medium  is  coagulated  sometimes  rapidly 
and  at  other  times  more  slowly.  In  the  first  sub-culture 
some  strains  of  the  bacillus  do  not  coagulate  milk  but 
on  further  sub-cultivation  they  quickly  acquire  this  property 
(Denys  and  Martin). 


SECTION  III.— BIOLOGICAL  PROPERTIES.  FIG.  224.— Pneumo- 

bacillus.    Stab  culture 

1.  Vitality    and    virulence. — Cultures    of   the    pneumo-  in  gelatin.  Tthday. 
bacillus   are   rapidly    killed    at    60°-80°  C.,   but    in    dry 

albuminous  matter  the  bacillus  is  much  more  resistant :  it  seems  to  retain 
both  its  virulence  and  its  vitality  for  a  long  time  in  water  and  soil.  The 
virulence  of  different  strains  of  the  pneumobacillus  is  subject  to  con- 
siderable variations  ;  it  is  possible  that  there  are  different  varieties  of  the 
organism  (the  Bacillus  lactis  aerogenes  would  be  one  of  these  varieties,  see 
p.  415). 

2.  Toxin. — Filtered  cultures  contain  a  toxin  which  is  fatal  to  rabbits  and 
produces  symptoms  of  paralysis.     Post  mortem  the  intestines  are  congested 
and  show  small  haemorrhages . 

3.  Bio-chemical   reactions.     Indol.     Nitrites.— In   a   neutral   3   per   cent, 
solution  of  peptone  the  pneumobacillus  does  not  produce  indol.     It  forms 
nitrites  out  of  nitrates. 

Fermentation  reactions. — The  pneumobacillus  ferments  glycerin,  and 
certain  of  the  carbohydrates,  viz. :  glucose,  galactose,  arabinose,  mannite, 
dulcite,  saccharose^  lactose,  maltose,  ramnose  and  dextrin,  but  is  without 
action  on  erythrite.  Frankland  has  described  a  strain  which  does  not 
ferment  glycerin. 

Grimbert  recommends  the  following  medium  for  the  study  of  the  fermentation 
reactions  : — 

Test  substance,  3  grams. 

Dry  peptone,    ...  2       „ 

Water,     -          -  100  c.c 

Calcium  carbonate,   -  Quantum  sufficit. 

The  formation  of  gas  is  naked-eye  evidence  of  fermentation.  If  the  calcium 
carbonate  be  omitted  and  litmus  solution  added  the  blue  colour  of  the  latter  is 
changed  to  red  during  the  fermentation.  Glycerin  is  more  slowly  broken  up. 

2D 


418  THE  PNEUMOBACILLUS 

SECTION  IV.— DETECTION,  ISOLATION  AND   IDENTIFICATION  OF 
THE  PNEUMOBACILLUS. 

I.  In  sputum. — (a)  Prepare  films  and  stain  with  carbol-thionin  or  carbol- 
gentian- violet.     Gram's  stain  must  also  of  course  be  used. 

(b)  Inoculate  a  mouse  with  a  trace  of  the  sputum. 

II.  In  blood,  pus,  etc. — Microscopical  examination  and  cultures,  supple- 
mented by  the  inoculation  of  a  mouse,  will  render  the  identification  of  the 
organism  easy. 

HI.  In  pseudo-membranous  sore  throats. — (a)  Scrapings  from  the  false 
membrane  should  be  stained  with  a  single  stain  and  by  Gram's  method  and 
examined1  microscopically. 

(6)  Cultures  should  be  sown  on  coagulated  serum  as  in  the  case  of  diphtheria. 
In  15-20  hours  fairly  large,  round,  greyish,  viscous  colonies  appear  which  can 
be  easily  recognized  with  the  naked  eye  and  under  the  microscope. 

IV.  In  water. — Adopt  the  method  of  cultivation  in  dilute  carbolic  acid 
or  on  peptone  salt  agar  (Chap.  LXV.).  After  two  or  three  passages,  pour 
gelatin  plates  on  which  the  round  raised  dull  white  colonies  of  the  pneumo- 
bacillus will  easily  be  recognized.  Sow  one  of  these  colonies  in  broth,  and 
after  48  hours'  incubation  test  the  virulence  of  the  culture  on  mice. 

Differential  diagnosis  from  the  pneumococcus.— The  pneumobacillus  is 
easily  differentiated  from  the  pneumococcus  by  its  cultural  characteristics 
and  by  the  fact  that  it  is  gram-negative. 

Differential  diagnosis  from  the  colon  bacillus.— In  water  examination  the 
pneumobacillus  is  likely  to  be  confused  with  the  colon  bacillus,  but  the 
mistake  may  easily  be  avoided  by  bearing  in  mind  the  following  points. 


PNEUMOBACILLUS  . 


COLON  BACILLUS. 


Absence  of  motility  in  broth  culture. 

Encapsulated.     The  capsule  is  very  well  marked  in 
fluids  and  tissues,  but  less  visible  in  cultures. 

No  indol  formation  in  peptone  water. 
Ferments  glycerin. 


Motile. 
Non-encapsulated. 


Forms  indol. 

Does  not  ferment   gly- 
cerin. 


The  bacillus  of  rhinoscleroma. 

The  bacillus  of  rhinoscleroma  was  discovered  by  V.  Frisch.  It  is  found  in  the 
nasal  and  pharyngeal  lesions  of  rhinoscleroma,  and  may  also  multiply  in  the  deeper 
tissues  of  the  nasal  fossae.  Rona  found  the  organism  in  pure  culture  in  the  enlarged 
sub-maxillary  glands  in  a  case  of  the  disease. 

The  bacillus  of  rhinoscleroma  is  very  similar  to  the  pneumobacillus  :  Netter  and 
Gunther  regard  them  as  varieties  of  the  same  species.  Their  biological  character- 
istics however  justify  their  being  regarded  as  different  organisms  (Paltauf  and 
Bertarelli). 

Microscopical  appearance. — In  sections  of  rhinoscleroma  nodules,  encapsulated 
cocco-bacilli  resembling  the  pneumobacillus  in  shape  and  size  are  seen  in  the  interior 
of  certain  very  large  cells  (cells  of  Mickulicz)  which  have  an  excentrically  placed 
crescent-shaped  nucleus.  The  fluid  of  the  nodules  does  not  appear  on  microscopical 
examination  to  contain  the  organism,  but  by  sowing  cultures  its  presence  can  be 
demonstrated. 


THE   BACILLUS   OF  RHINOSCLEROMA  419 

Cultures. — The  bacillus  of  rhinoscleroma  grows  on  all  the  ordinary  labora- 
tory media.  Unlike  the  pneumobacillus  it  does  not  grow  on  slightly  acid 
media,  and  does  not  ferment  carbohydrates  ;  further,  its  cultures  are  much 
more  scanty  than  those  of  the  pneumobacillus  (Paltauf). 

In  cultures,  the  bacillus  of  rhinoscleroma  always  forms  capsules  which  may 
be  easily  demonstrated  by /Hie  following  method.  Dilute  a  little  of  the 
growth  in  a  1  per  cent,  solution  of  acetic  acid,  spread  on  a  slide,  dry,  and 
stain  with  aniline-violet :  examine  in  water. 

On  broth,  agar  and  serum — The  growth  on  these  media  is  very  similar  to 
the  growth  of  the  pneumobacillus  but  more  scanty. 

On  gelatin  the  growth  is  thread-like  and  very  limited.  The  tylotate 
appearance  so  characteristic  of  the  pneumobacillus  is  never  produced. 

On  milk.     The  medium  is  not  coagulated. 

Experimental  inoculation.— Laboratory  animals  are  not  susceptible  to 
inoculation  with  the  bacillus  of  rhinoscleroma. 

Lowenberg's  bacillus. 

(The  bacillus  of  ozcena.) 

The  bacillus  found  in  the  mucous  exudates  in  ozaena  by  Lowenberg  and  Abel 
is  no  longer  regarded  as  the  cause  of  ozsena.  It  resembles  the  pneumobacillus  so 
closely  that  it  seems  necessary  to  regard  the  two  organisms  as  identical  (Viollet, 
de  Simoni,  and  others). 

The  microscopical  appearance,  the  cultural  characteristics  and  the  results  of  inocula- 
tion are  the  same  in  both  cases.  The  only  differences  between  them  seem  to  be  that 
the  bacillus  of  ozsena  does  not  ferment  all  the  carbohydrates  which  are  fermented 
by  the  pneumobacillus  and  does  not  coagulate  milk. 


« I 


CHAPTER   XXV. 

THE    PAEATYPHOID    BACILLI.1 
(THE  PARACOLON  BACILLI.) 

The  origin  of  the  terms  paratyphoid  and  paracolon,  p.  420.  The  relation  of  the 
"  paratyphoid  "  bacilli  to  the  "  hsemorrhagic  septicaemia  "  group  of  organisms  and  to 
the  "  enteritidis  "  group,  p.  421. 

The  origin  and  definition  of  the  "  Salmonella  group,"  p.  422. 

Other  names  suggested  for  the  "  paratyphoid  group,"  p.  422. 

The  classification  adopted  in  the  following  pages,  p.  422. 

UNDER  the  heading  of  paratyphoid  bacilli  are  described  certain  organisms 
which  in  many  respects  resemble  the  typhoid  and  the  colon  bacilli  :  they 
are  all  gram-negative  motile  bacilli  which  do  not  form  spores  and  do  not 
liquefy  gelatin.  From  the  clinical  standpoint  also,  though  the  symptoms 
are  markedly  different,  there  is  a  certain  resemblance  in  that  the  diseases 
produced  by  the  typhoid  and  paratyphoid  bacilli  and  probably  also  by 
the  colon  bacillus  are  all  primarily  of  a  septicsemic  nature. 

The  name  paratyphoid  was  introduced  by  Achard  and  Bensaude  in  1896 
to  describe  an  organism  (paratyphoid  B)2  resembling  the  typhoid  bacillus, 
which  they  had  isolated  from  a  case  of  osteomyelitis  following  an  attack  of 
a  disease  clinically  indistinguishable  from  enteric  fever,  and  from  the  urine 
of  another  case  of  a  similar  disease. 

In  1897  Besson  isolated  a  similar  organism  from  a  case  of  pericarditis  follow- 
ing a  disease  which  had  been  diagnosed  as  enteric  fever. 

In  1897  Widal  and  Nobecourt  also  found  a  similar  organism  (paratyphoid 
B)2  in  pus  from  a  thyroid  abscess  in  which  there  were  no  symptoms  of  a  general 
infection.  To  this  organism  they  gave  the  name  "  para-colon  "  bacillus. 

Gwyn  in  1898  was  the  first  to  isolate  a  "  para-colon  "  bacillus  from  the 
blood  of  a  person  suffering  fro,m  a  disease  clinically  indistinguishable  from 
enteric  fever. 

In  1900-1  Schottmuller  undertook  an  investigation  into  the  nature  of  the 
organisms  present  in  the  blood  of  cases  which  had  the  clinical  symptoms  of 
enteric  fever.  In  addition  to  the  typhoid  bacillus  he  found  two  other  species 
of  organisms,  closely  related  to  the  typhoid  bacillus  and  to  each  other,  to 
which  Brion  and  Kayser  gave  the  names  paratyphoid  A  and  paratyphoid  B  ; 
the  latter  being  the  more  frequently  found.  To  these  organisms  then  the 
term  "  paratyphoid  "  is  properly  applied  :  organisms,  that  is,  which  have  many 

1  This  part  of  the  subject  has  been  entirely  rewritten. 

2  See  Boycott,  Journal  of  Hygiene,  vi.  33  et  seq. 


THE  PARATYPHOID  BACILLI  421 

of  the  bacteriological  characteristics  of  the  typhoid  and  colon  bacilli  and  which 
give  rise  to  a  clinical  disease  having  all  the  symptoms  of  enteric  fever. 

Further  study  however  soon  revealed  the  fact  that  these  paratyphoid  bacilli 
were,  at  all  events  in  the  laboratory,  very  closely  related  to  if  not  identical 
with  organisms  which  had  been  isolated  from  certain  septicaemic  diseases  in 
animals  accompanied  by  haemorrhages — the  hsemorrhagic  septicaemia  group. 
The  first  of  this  group  to  be  described  was  that  isolated  by  Salmon  and 
Theobald  Smith  in  1885  from  swine  suffering  from  hog-cholera  and  known  as 
the  bacillus  of  hog-cholera.1 

The  paratyphoid  bacilli  especially  the  B  variety,  had  also  many  charac- 
teristics in  common  with  an  organism  isolated  by  Gaertner  in  1888  at  Franken- 
hausen  from  an  epidemic  of  food-poisoning,  and  known  as  the  bacillus 
enteritidis  Gaertner. 

Closely  related  also  to  the  paratyphoid  bacilli  is  an  organism  known  as 
the  bacillus  enteritidis  Aertrycke,2  isolated  in  1898  by  de  Nobele  from  an 
epidemic  of  food-poisoning  at  Aertrycke  in  Belgium  and  by  Durham  at 
Hatton  in  England.  By  its  cultural  characteristics  this  organism  cannot  be 
distinguished  from  the  bacillus  enteritidis  Gaertner?  but  as  Durham  showed 
by  an  application  of  the  agglutination  reaction,  then  recently  introduced, 
the  two  could  be  sharply  differentiated . 

Hence  in  the  first  quinquennium  of  the  century  a  number  of  organisms 
were  known  which  from  the  laboratory  point  of  view  were  all  very  like  each 
other,  but  which — and  this  seemed  remarkable— gave  rise  to  different  dis- 
eases. The  paratyphoid  bacilli  A  and  B  caused  a  septicaemic  disease  clinically 
almost  if  not  quite  identical  with  enteric  fever  ;  the  haemorrhagic  septicaemia 
group  caused  a  septicaemia  and  diarrhoea  in  animals  ;  while  the  group  consist- 
ing of  Gaertner's  and  Durham's  and  de  Nobele's  bacilli  were — and  still  are — 
regarded  as  the  cause  of  epidemics  of  food-poisoning. 

It  was  easy  to  distinguish  the  paratyphoid  A  bacillus  from  the  other  bacilli 
mentioned  both  by  its  cultural  characteristics  and  by  its  agglutination 
reactions.  The  gaertner  bacillus  also  could  by  its  agglutination  reactions 
be  distinguished  from  the  aertrycke  bacillus,  the  bacillus  of  hog-cholera  and 
the  bacillus  paratyphoid  B. 

Then  difficulties  arose  as  to  the  nature  of  the  three  last-named  organisms. 
The  bacillus  of  hog-cholera  was  soon  shown  to  be  identical  with  the  aertrycke 
bacillus ;  and  the  relationship  of  the  latter  bacillus  to  the  paratyphoid 
B  bacillus  therefore  alone  remained  to  be  determined.  The  position  in  1906 
was  summarized  by  Boycott :  "  On  the  whole,  the  distinction  between 
hog-cholera  [aertrycke]  and  paratyphoid  B,  though  slender,  seems  to  be 
real.  The  morbific  relations  to  man  are  different,  for  while  the  former  gives 
rise  to  a  sudden  acute  illness  (food-poisoning),  paratyphoid  B  causes  a  disease 
with  no  clear  clinical  distinctions  from  enteric  fever."  4 

With  a  view  to  studying  Castellani's  absorption  reaction  Bainbridge  took 
the  paratyphoid  bacilli  as  a  suitable  group  upon  which  to  work.  By  the 
aid  of  this  reaction  he  has  now  made  it  clear  that  the  bacillus  paratyphoid  B 

1  At  the  time,  these  authorities  believed  the  organism  to  be  the  cause  of  hog-cholera 
and  their  opinion  was  accepted  by  other  observers  subsequently.     Hence  the  name  by 
which  it  is  still  very  commonly  known,  the  bacillus  of  hog-cholera,  bacillus  suipestifer,  or 
bacillus  cholerce  suis.     In  1903  however  the  researches  of  de  Schweintz,  Dorset  and  others 
showed  that  hog-cholera  is  not  to  be  ascribed  to  the  Salmon-Smith  bacillus  but  to  a 
filter-passing  organism  (Chap.  LXIV.),  the  hog-cholera  bacillus  being  merely  a  secondary 
infection. 

2  This  bacillus  will  in  future  be  described  as  "  the  aertrycke  bacillus." 

3  In  future  referred  to  as   ''•  the  gaertner  bacillus." 

4  See  however  Paratyphoid  B  as  a  cause  of  food -poisoning  p.  432. 


422 


THE  PARATYPHOID   BACILLI 


is  separate  and  distinct  from  the  aertrycke  bacillus,  and  this  observation  has 
been  confirmed  by  Dean  from  a  study  of  complement  fixation  reactions. 

Bainbridge  has  further  shown  that  unless  the  absorption  tests  be  applied  the 
paratyphoid  B  bacillus  cannot  be  differentiated  from  the  aertrycke  bacillus, 
and  that  in  practically  all  cases  the  so-called  paratyphoid  B  bacillus  isolated 
from  cases  of  food-poisoning  is  in  reality  the  aertrycke  bacillus.  The  para- 
typhoid B  bacillus  can  however  give  rise  to  acute  gastro-enteritis  though  this 
would  at  present  seem  to  be  a  very  uncommon  association  (p.  432). 

Bainbridge's  investigations  have  also  demonstrated  that  a  number  of  the 
bacteria  causing  diseases  in  the  lower  animals  (vide  post)  are  not  separate 
species,  but  are  either  identical  with  the  paratyphoid  B  bacillus,  the 
aertrycke  bacillus  or  the  gaertner  bacillus,  or  are  impure  cultures  of  two  or 
more  of  these  organisms.  The  various  rat  and  mice  viruses  are  therefore 
shown  by  laboratory  procedures,  as  well  as  by  practical  experience,  not  to 
be  so  harmless  to  man  and  the  domestic  animals  as  they  are  claimed  to  be. 

Lignieres  proposed  to  designate  all  those  organisms  which  had  the  morpho- 
logical and  cultural  attributes  of  the  bacillus  of  hog-cholera  [bacillus  aertrycke} 
by  the  name  Salmonella  after  Salmon  to  whom  the  discovery  of  that  organism 
is  due.  This  term  has  met  with  some  acceptance  on  the  Continent,  and  is  a 
convenient  appellation  under  which  to  include  a  number  of  organisms  very 
closely  related  bacteriologically,  though  clinically  the  diseases  to  which  they 
give  rise  generally  differ.  It  forms  an  appropriate  classification  for  purposes 
of  practical  bacteriology  and  will  therefore  be  adopted  here.  The  Salmonella 
group,  used  in  its  original  sense  as  defined  above,  includes  the  following 
organisms  :  the  bacillus  paratyphosus  B,  bacillus  enteritidis  Aertrycke  (syn. 
bacillus  suipestifer},  and  bacillus  enteritidis  Gaertner  :  as  well  as  a  number 
of  organisms  which  have  received  specific  names  but  which  have  now  been 
shown  to  be  identical  with  one  or  other  of  the  preceding  :  these  are  bacillus 
danysz,  bacillus  typhi  murium,  bacillus  psittacosis  and  bacillus  icteroides. 

Other  names  also  have  been  proposed  for  the  group  of  organisms  discussed 
in  this  chapter :  Theobald  Smith  suggested  "  the  hog-cholera  group  " ;  Durham, 
the  "  intermediate  group,"  and  Trautmann,  the  "paratyphoid  group." 

The  paratyphoid  A  bacillus  is  obviously  excluded  on  cultural  grounds  from 
the  Salmonella  group.  The  "paratyphoid  bacilli"  will  therefore  be  dealt  with 
under  two  headings  (1)  The  bacillus  paratyphosus  A  (2)  The  Salmonella  group. 

The  paratyphoid  bacilli  may  then  be  grouped  thus — 


GROUP. 

SPECIFIC  ORGANISMS. 

SYNONYMS. 

I. 
II. 

Bacillus  paratyphosus  A. 
The  Salmonella  group  :  — 
(i)  Bacillus  paratyphosus  B. 
(ii)  Bacillus  enteritidis  Aertrycke. 
(iii)  Bacillus  enteritidis  Gaertner. 

(  Bacillus  of  hog  cholera. 
-  B.  suipestifer. 
B.  cholerse  suis. 

The  organisms  known  as  Bacillus  danysz,  B.  typhi  murium,  B.  psitta- 
cosis and  B.  icteroides  are  either  identical  with  one  or  other  of  the 
members  of  the  Salmonella  group  or  are  mixed  cultures  of  two  or 
more  of  these  organisms. 

CHAPTER  XXVI.1 

BACILLUS  PARATYPHOSUS  A. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  424. 

Section  II. — Morphology,  p.  424. 

1.  Microscopical  appearance  and  staining  reactions,  p.  424.  2.  Cultural  character- 
istics, p.  424. 

Section  III. — Biological  properties,  p.  424. 

1.  Biochemical  reactions,  p.  424.  2.  Virulence,  p.  425.  3.  Toxin,  p.  425.  4  Vac- 
cination, p.  425.  5.  Agglutination,  p.  426.  6.  Absorption  tests,  p.  427.  7.  Com- 
plement fixation,  p.  428. 

Section  IV. — The  diagnosis  of  paratyphoid  A  infections.     The  isolation  and  identification 
of  the  bacillus,  p.  428. 

The  pseudo-paratyphoid  A  bacillus,  p.  430. 

THE  paratyphoid  A  bacillus  was  first  described  by  Schottmiiller  who 
isolated  it  from  the  blood  of  patients  suffering  from  a  disease  clinically 
indistinguishable  from  enteric  fever. 

Paratyphoid  A  fever  is  a  septicaemia  characterized  by  "  a  mild  pyrexia  simulating 
enteric  fever,  marked  by  no  acute  gastric  or  intestinal  symptoms  and  rarely  fatal " 
(Firth).  The  lymphatic  system  is  less  affected  than  in  enteric  fever  though  one 
case  is  recorded  where  a  single  perforation  was  found  (Grattan  and  Wood). 

The  bacillus  has  never  been  isolated  in  England  (Bainbridge)2  and  com- 
paratively few  cases  of  paratyphoid  A  infection  have  been  recorded  on  the 
Continent  of  Europe.  In  America  its  distribution  is  uncertain  :  but  it  is 
worth  noting  that  in  one  year  in  the  Allegheny  General  Hospital  the  relation 
of  paratyphoid  A  fever  (48  cases)  to  enteric  fever  was  8  to  11  (Proescher  and 
Roddy). 

In  India,  on  the  other  hand,  paratyphoid  A  fever  is  very  prevalent. 
Grattan  and  Wood  estimate  that  one-third  of  the  cases  of  "  simple  continued 
fever  "  in  India  are  cases  of  paratyphoid  A  fever,  and  so  constantly  is  the 
A  variety  of  the  bacillus  found  that  paratyphoid  fever  in  that  country  connotes 
an  infection  with  the  paratyphoid  A  bacillus  (Firth). 

The  bacillus  apparently  remains  in  the  system  for  a  time  after  an  attack  of  para- 
typhoid A  fever  and  "  carriers  "  would  appear  to  be  the  chief  agent  in  the  dissemina- 
tion of  the  disease  (Firth).  Convalescents  are  usually  infective  for  a  comparatively 
short  period,  and  "  chronic  carriers  "  (persons  in  whom  the  bacillus  remains  more 
than  3  months),  would  seem  to  be  uncommon. 

1  This  chapter  has  been  rewritten. 

2  Bainbridge,  F.  A.,  The  Milroy  Lectures,  Royal  College  of  Physicians,  Lancet,  1912,  i. 


424  THE  PARATYPHOID  A.   BACILLUS 

The  paratyphoid  A  bacillus  has  been  recovered  from  the  gall  bladder  after  death 
and  during  operations  for  gall  stones  or  cholecystitis  ;  it  has  also  been  isolated  once 
from  an  abdominal  abscess  and  once  from  an  apparently  healthy  man.  Bainbridge 
states  that  it  has  been  isolated  from  a  case  of  acute  enteritis.  The  organism  has 
never  yet  been  recovered  from  other  than  human  tissues. 


SECTION  I.— EXPERIMENTAL   INOCULATION. 

All  strains  of  the  paratyphoid  A  bacillus  are  virulent  for  laboratory  animals. 
The  inoculation  of  4  c.c.  of  a  broth  culture  sub-cutaneously  is  fatal  to  guinea- 
pigs  (Brion  and  Kayser)  :  in  mice,  inoculation  produces  a  fatal  disease 
accompanied  with  symptoms  of  acute  enteritis. 

Guinea-pigs  and  mice  are  easily  infected  with  a  fatal  disease  by  feeding 
them  on  cultures  of  the  bacillus. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  paratyphoid  A  bacillus  is  a  short  stout  rod-shaped  organism  with 
rounded  ends  often  having  the  appearance  of  a  cocco-bacillus  :  in  old  cultures 
long  filamentous  forms  are  occasionally  seen.  It  is  very  motile  and  is  provided 
with,  from  four  to  ten  delicate  flagella.  Morphologically  it  is  indistinguishable 
from  the  other  bacilli  of  the  typhoid-colon  group. 

Staining  reactions. — The  paratyphoid  A  bacillus  stains  readily  with  the 
ordinary  basic  aniline  dyes  and  occasionally  exhibits  polar  staining.  The 
bacillus  is  decolourized  by  Gram's  method. 

2.  Cultural  characteristics. 

The  paratyphoid  A  bacillus  is  a  facultative  aerobe  and  grows  readily  on 
the  ordinary  media  in  a  manner  very  like  the  typhoid  bacillus. 

Broth. — The  medium  is  rendered  cloudy  and  has  a  watered  silk  appearance. 

Gelatin. — The  colonies  are  iceberg-like,  translucent  and  bluish  :  in  stroke 
culture  the  growth  is  thin  and  streaked  with  blue.  The  medium  is  not 
liquefied. 

Potato. — On  potato  the  bacillus  gives  a  barely  visible  glaze. 

Artichoke. — Generally  colourless  :  a  green  colour  may  be  produced  after 
some  time. 

Milk. — Milk  is  not  coagulated. 

Litmus  milk. — In  litmus  milk,  acid  is  formed,  the  colour  of  the  litmus 
being  changed  to  pink  (p.  373) :  the  acidity  is  permanent.  No  clot  is  formed. 
The  'permanent  acidity  without  clot  is  peculiar  to  this  member  of  the  typhoid- 
colon  group. 

Litmus  whey. — A  slight  but  permanent  acidity  indicated  by  the  change 
in  colour  of  the  litmus  from  amethyst  to  pink. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 

1.  Biochemical  reactions. 

(a)  Fermentation  of  carbohydrates.— The  paratyphoid  A  bacillus  produces 
acid  and  gas  in  glucose  but  has  no  action  (producing  neither  acid  nor  gas) 
on  lactose.  The  bacillus  is  thus  easily  differentiated  from  the  typhoid  bacillus 
on  the  one  hand  and  from  the  colon  bacillus  on  the  other. 


BIOLOGICAL  PROPERTIES  425 

It  forms  acid  and  gas  also  in  Isevulose,  maltose,1  galactose,  mannite,  dulcite, 
sorbite  and  glycerin  :  but  neither  acid  nor  gas  in  raffinose,  saccharose  and 
lactose. 

The  paratyphoid  A  bacillus  does  not  ferment  carbohydrates  so  power- 
fully as  the  paratyphoid  B  bacillus. 

(/:?)  Neutral-red  media. — The  paratyphoid  A  bacillus  like  the  colon  bacillus 
reduces  neutral-red,  and  in  media  containing  the  dye  may  give  rise  to  a 
greenish  fluorescence,  but  the  reaction  is  less  marked  than  with  the  colon 
bacillus  and  the  Salmonella  group.  Fluorescence  in  neutral-red  media  is 
however  a  very  inconstant  change  ;  the  best  medium  for  the  reaction  is  agar 
containing  1  per  cent,  of  glucose  and  1  c.c.  per  litre  of  a  saturated  aqueous 
solution  of  neutral-red. 

(7)  Endo's  medium. — On  fuchsin-agar  decolourized  with  sodium  sulphite 
the  bacillus,  like  the  typhoid  bacillus,  gives  colourless  colonies. 

(8)  Caffeine  media. — According  to  Ducamp  the   paratyphoid  A  bacillus 
will  not  grow  in  broth  containing  O5  per  cent,  of  caffeine  (vide  B.  para- 
typhosus  B}. 

(«)  Malachite  green  media. — Malachite  green  is  decolourized  by  the  para- 
typhoid A  bacillus  (1  week)  but  more  slowly  than  by  the  bacilli  of  the 
Salmonella  group  (48  hours). 

(f)  Vaccinated  media. — The  paratyphoid  A  bacillus  does  not  grow  on 
media  which  have  already  served  for  the  growth  of  the  typhoid,  colon, 
paratyphoid  A  or  paratyphoid  B  bacilli. 

(r?)  Indol. — The  paratyphoid  A  bacillus  forms  no  indol  in  culture. 

2.  Virulence. 

Sacquepee  and  Chevrel  were  able  to  increase  the  virulence  of  the  bacillus 
by  passing  it  through  a  series  of  animals  by  sub-cutaneous  inoculation.  The 
inoculation  of  O5  c.c.  of  a  twenty-four-hour  culture  of  the  exalted  virus  in 
broth  was  sufficient  to  kill  guinea-pigs. 

The  virulence  is  lost  somewhat  readily  in  culture. 

3.  Toxin. 

The  paratyphoid  A  bacillus  produces  a  soluble  toxin  in  culture  media. 
Cultures  sterilized  at  60°  C.  are  pyogenic  when  inoculated  beneath  the  skin 
of  guinea-pigs.  Cultures  of  an  exalted  virus  sterilized  by  heat  or  filtered 
through  porcelain  kill  guinea-pigs  when  inoculated  sub-cutaneously  in  doses 
of  3-10  c.c. 

4.  Vaccination. 

Guinea-pigs  and  white  rats  can  be  easily  vaccinated  against  the  para- 
typhoid A  bacillus  by  inoculating  them  with  attenuated  or  sterilized  cultures. 
From  the  experiments  of  Gushing  and  of  Sacquepee  and  Chevrel  it  would 
appear  that  animals  immunized  against  the  paratyphoid  A  bacillus  are  also 
immunized  but  to  a  lesser  degree  against  the  typhoid  bacillus  (intervaccina- 
tion  or  group  immunization).  The  serum  of  immunized  animals  is  distinctly 
immunizing  and  bacteriolytic  for  paratyphoid  A. 

Human  vaccination. — In  man,  vaccination  with  Wright's  typhoid  vaccine 
affords  no  immunity  against  an  infection  with  the  paratyphoid  A  bacillus. 

Prophylactic  vaccination  of  the  human  subject  though  suggested  (Bain- 
bridge,  Leishman)  has  not  yet  been  attempted.  Certain  preliminary 
laboratory  experiments  have  however  been  quite  recently  recorded  by 
Cummins  and  Gumming. 

1  The  amount  of  gas  formed  out  of  maltose  is  always  small  whatever  the  organism. 


426 


THE  PARATYPHOID  A.   BACILLUS 


5.  Agglutination. 

The  serum  of  vaccinated  animals  and  of  persons  suffering  from  paratyphoid 
A  fever  will  agglutinate  the  paratyphoid  A  bacillus.  The  serum-diagnosis 
of  paratyphoid  A  fever  however  requires  considerable  skill  and  care  on  the 
part  of  the  observer. 

Agglutination  with  the  serum  of  immunized  animals. — The  serum  of  animals 
highly  immunized  against  the  paratyphoid  A  bacillus  contains  both  specific 
agglutinins  and  group  agglutinins.  Not  only  does  such  a  serum  agglutinate 
the  paratyphoid  A  bacillus  but  it  agglutinates  also  the  typhoid  bacillus  and 
other  related  bacilli ;  but  if  the  limits  of  agglutination  be  determined  it  will 
be  found  that  the  serum  agglutinates  the  paratyphoid  A  bacillus  in  a  much 
higher  dilution  than  it  agglutinates  the  typhoid  or  any  other  related  bacillus. 

Thus  an  anti-paratyphoid  A  serum  agglutinates  all  strains  of  the  para- 
typhoid A  bacillus  in  dilutions  of  1-1,000,  1-5,000  and  even  1-40,000.  On 
the  other  hand  it  has  very  little  agglutinating  action  on  strains  of  the  para- 
typhoid B  bacillus,  on  the  gaertner  bacillus,  or  on  the  aertrycke  bacillus 
and  only  agglutinates  the  typhoid  bacillus  in  dilutions  of  1-200,  1-100 
or  1-20. 

The  following  table l  illustrates  this  : — 

Agglutination  limits  after  incubation  for  2  hours  at  42°  C.  Macroscopic  method. 
In  all  cases  control  tubes  showed  no  agglutination. 


SERUM. 

EMULSION  OF  BACILLI. 

Paratyphoid  A. 

Paratyphoid  A. 

Paratyphoid  B. 

Aertrycke. 

Gaertner. 

50,000 

<  100 

<  100 

<  100 

Conversely,  experimental  typhoid  serums  have  little  action  on  strains 
of  the  paratyphoid  A  bacillus  and  only  agglutinate  them  in  low  dilution. 

A  similar  statement  is  justifiable  for  paratyphoid  B,  aertrycke  and  gaertner 
serums. 

To  sum  up  :  By  means  of  the  agglutination  reaction  with  an  experimental 
serum  the  paratyphoid  A  bacillus  can  with  certainty  be  identified,  being  clearly 
differentiated  from  the  typhoid,  paratyphoid  B,  gaertner  and  aertrycke  bacilli. 

Agglutination  reaction  with  human  serum. — The  conditions  are  somewhat 
different  when  working  with  an  human  serum. 

The  experience  in  cases  of  paratyphoid  fever  in  India  is  as  follows  : — The 
agglutination  titre  is  usually  low  (1-20  to  1-40)  and  the  reaction  commonly 
transient :  it  may  be  quite  as  high  for  the  typhoid  bacillus  as  during  an  ordinary 
attack  of  enteric  fever  and  the  co-agglutinin  for  this  bacillus  may  remain  after 
the  specific  agglutinin  has  vanished.  Rarely,  the  specific  agglutinin  alone  is 
present ;  and  sometimes  both  it  and  the  co-agglutinin  for  the  typhoid  bacillus 
are  present  in  so  small  an  amount  and  for  so  short  a  time  as  to  be  easily  over- 
looked. Finally  it  is  possible  in  undoubted  cases  of  paratyphoid  A  fever 
for  the  serum  to  contain  co-agglutinins  for  the  typhoid  and  paratyphoid  B 
bacilli  but  no  specific  agglutinin  (Firth).2 

The  group  agglutinin  for  the  typhoid  bacillus  quickly  disappears  in  para- 

1  Bainbridge,  F.  A.,  Journal  of  Pathology  and  Bacteriology,  xiii.   p.  341. 

2  Journal  of  the  Royal  Army  Medical  Corps. 


ABSORPTION  OF  AGGLUTININS  427 

typhoid  A  fever,  in  contrast  to  the  persistence  of  the  specific  agglutinin  which 
follows  enteric  fever  (Firth). 

Moreover,  Grattan  and  Wood l  record  that  "  In  some  cases  the  limits  of 
co-agglutination  for  the  typhoid  bacillus  exceeded  the  limits  of  specific 
agglutination  :  and  that  in  other  cases  again,  at  one  period  of  the  disease  the 
limits  of  co-agglutination  for  the  typhoid  bacillus  exceeded  the  limits  of 
specific  agglutination,  and  at  another  period  the  limits  of  specific  agglutination 
exceeded  the  limits  of  co-agglutination." 

These  observers  find  that  "  antityphoid  inoculation  seldom  if  ever  pro- 
duces co-agglutinins  for  the  paratyphoid  A  bacillus.  Hence  in  a  typhoid- 
vaccinated  individual  a  serum  reaction  against  the  paratyphoid  A  bacillus 
in  a  dilution  of  1-20  is  strong  evidence  of  paratyphoid  A  fever.  "  And  in 
inoculated  persons  an  attack  of  paratyphoid  A  fever  raises  the  titre  of 
agglutination  for  the  typhoid  bacillus  about  the  8th  day,  while  the  agglutinins 
for  the  paratyphoid  A  bacillus  do  not  appear  much  before  the  12th  day." 

6.  Absorption  tests. 

To  explain  the  phenomena  just  described  it  is  necessary  to  assume  the 
presence  of  group  agglutinins,  or,  in  those  cases  where  co -agglutination  is 
very  marked,  the  existence  of  a  double  infection. 

Castellani's  method  of  absorption  of  agglutinins  may  be  used  for  diagnostic 
purposes  when  the  results  of  the  agglutination  tests  are  doubtful. 

Let  us  take  the  case  of  a  serum  which  has  but  little  agglutinating  action  on  the 
typhoid  bacillus  but  agglutinates  the  paratyphoid  A  bacillus  in  higher  dilution 
(1—40  to  1—100).  To  such  a  serum  add  paratyphoid  A  bacilli  in  sufficient  quantity 
to  remove  the  whole  of  the  paratyphoid  A  agglutinins  and  centrifuge.  Then  test 
the  agglutinating  action  of  the  clear  supernatant  fluid  on  both  the  typhoid  and 
paratyphoid  A  bacilli  (to  ascertain  that  the  agglutinins  for  the  paratyphoid  A 
bacillus  have  been  removed).  If  the  typhoid  bacillus  be  agglutinated  it  may  be 
assumed  that  agglutinins  were  present  in  the  serum  for  both  the  typhoid  and  para- 
typhoid A  bacilli ;  but  if  on  the  other  hand  the  typhoid  bacillus  is  not  agglutinated, 
then  the  original  action  of  the  serum  on  the  typhoid  bacillus  was  due  to  the  presence 
in  it  of  a  co-agglutinin.  But  in  cases  of  paratyphoid  A  fever,  when  the  serum  agglu- 
tinates both  the  typhoid  and  paratyphoid  A  bacilli,  absorption  with  the  paratyphoid 
A  bacillus  removes  all  agglutinins — both  specific  and  group ;  while  absorption  with 
the  typhoid  bacillus  removes  merely  the  co-agglutinin  for  the  typhoid  bacillus, 
leaving  the  specific  agglutinin  for  the  paratyphoid  A  bacillus  intact  even  though 
it  mav  in  the  first  instance  have  only  been  demonstrable  in  a  dilution  of  1-20  (Harvey 
and  Wood). 

If  a  person  inoculated  against  enteric  fever  and  whose  serum  agglutinates 
the  typhoid  bacillus  become  infected  with  a  paratyphoid  A  infection,  absorp- 
tion of  the  serum  with  the  paratyphoid  A  bacillus  only  reduces  the  titre  and 
does  not  entirely  remove  the  agglutinins  specific  for  the  typhoid  bacillus 
(Harvey  and  Wood). 

If  in  a  case  of  paratyphoid  A  fever  the  serum  agglutinate  the  typhoid 
bacillus  but  not  the  paratyphoid  A  bacillus,  absorption  with  the  latter  will 
remove  the  whole  of  the  agglutinin  (co-agglutinin)  for  the  former,  whereas 
the  agglutinins  for  the  typhoid  bacillus  in  cases  of  enteric  fever  are  not 
removed  by  absorption  with  the  paratyphoid  A  bacillus  (Harvey  and  Wood). 

Another  difference  between  the  specific  and  group  agglutinins  for  the 
paratyphoid  A  bacillus  is  that  when  the  serum-emulsion  mixture  is  put  up 
in  sedimentation  tubes  the  specific  agglutination  appears  almost  immediately 
while  the  co-agglutination  does  not  appear  for  some  hours  (Harvey  and  Wood). 

To  sum  up  :   The  serum  of  persons  suffering  from  paratyphoid  A  fever  usually 

1  Journal  of  the  Royal  Army  Medical  Corps. 


428  THE  PARATYPHOID  A.   BACILLUS 

agglutinates  the  paratyphoid  A  bacillus  only  in  low  dilution  (1-20  to  1-40). 
Co-agglutinins  for  related  organisms  and  especially  for  the  typhoid  bacillus  are 
as  a  rule  also  present  in  the  serum  :  the  amount  of  the  co-agglutinin  is  very 
variable  often  exceeding  the  titre  of  the  specific  agglutinin,  and  moreover  may 
be  present  to  the  exclusion  of  the  latter.  Absorption  tests  will  alloiv  of  a  correct 


7.  Complement  fixation. 

The  serum  of  animals  immunized  with  the  paratyphoid  A  bacillus  contains 
an  immune  body  which  is  fixed  by  both  the  paratyphoid  A  and  typhoid  bacilli, 
but  the  converse  does  not  hold  good  ;  the  immune  body  in  the  serum  of  animals 
immunized  with  the  typhoid  bacillus  is  not  fixed  by  the  paratyphoid  A 
bacillus  (Rieux  and  Sacquepee). 

In  healthy  men  inoculated  against  enteric  fever,  specific  amboceptors  for 
the  paratyphoid  A  bacillus  were  present  in  amounts  apparently  equal  to  the 
specific  amboceptors  for  the  typhoid  bacillus,  though  in  no  case  had  the 
individuals  tested  suffered  from  paratyphoid  A  fever  (Grattan  and  Wood). 

If  the  dosage  of  antigen  and  antibody  be  carefully  determined,  the  para- 
typhoid A  bacillus  can  be  clearly  and  absolutely  differentiated  from  the 
typhoid  and  paratyphoid  B  bacilli  by  the  complement  fixation  method 
(H.  R.  Dean). 

Dean's  method.  Preparation  of  extract. — Agar  cultures  were  sown  in  Roux  bottles, 
incubated  at  37°  C.  for  48  hours  and  then  emulsified  in  20  c.c.  of  distilled  water. 
The  emulsion  was  tubed  in  quantities  of  5  c.c.,  and  in  some  cases  heated  to  60°  C. 
(this  heating  had  no  effect  on  the  properties  of  the  extract) ;  then  after  being 
thoroughly  shaken  it  was  frozen  hard,  and  subsequently  thawed  slowly  at  room 
temperature.  After  freezing  and  thawing  twice  the  emulsion  was  shaken  all  night 
in  a  shaking  machine,  again  frozen  and  thawed  twice,  shaken  again  all  night  and 
then  centrifuged  until  the  supernatant  liquid  was  clear  or  only  slightly  opalescent. 
The  extract  was  then  stored  in  the  cold  room. 

Antiserum. — Rabbits  were  inoculated  intra-venously  four  or  five  times  at  intervals 
of  4  or  5  days  with  saline  emulsions  of  24-hour  agar  cultures.  The  animals  were 
tested  on  the  eighth  day  after  the  last  inoculation  and  if  the  serum  was  satisfactory 
were  bled  on  the  ninth  or  tenth  day.  .The  serum  was  heated  at  56°  C.  for  half  an 
hour  and  stored  in  quantities  of  2  c.c. 

Complement. — The  guinea-pig  serum  was  prepared  on  the  day  of  use. 

Hsemolytic  system. — Sheep  red  cells  and  rabbit-sheep  serum. 

Experimental  data. — The  bulk  of  fluid  in  each  tube  was  2 '5  c.c. — 0'5  c.c.  of  diluted 
bacterial  extract  (antigen),  0'5  c.c.  of  diluted  serum  (antibody)  and  0'5  c.c.  of  a  1  in 
10  dilution  of  fresh  guinea-pig  serum  (complement).  After  incubation  there  were 
added  0'5  c.c.  of  the  dilution  of  haemolytic  serum  (determined  on  the  day  of  the 
experiment)  and  0'5  c.c.  of  a  1  in  20  suspension  of  washed  sheep  cells. 

The  dilution  of  antiserum  necessary  for  a  differentiation  experiment  can  be 
ascertained  by  the  titration  of  the  antiserum  with  the  homologous  extract.  As  a 
rule  satisfactory  differentiation  can  be  obtained  with  the  greatest  dilution  of  anti- 
serum  which  is  found  to  give  a  thoroughly  satisfactory  reaction  with  the  homologous 
extract. 

With  low  dilutions  of  antiserum  a  marked  group  reaction  is  obtained  and  dif- 
ferentiation is  impossible.  The  group  antibodies  can  however  be  removed  by 
absorption  (vide  supra). 


SECTION  IV.— THE  DIAGNOSIS  OF  PARATYPHOID  A.  INFECTIONS. 

The  isolation  and  identification  of  the  bacillus. 

The  diagnosis  of  a  paratyphoid  A  infection  should  be  based  upon  the 
demonstration  of  the  specific  organism  in  the  tissues  or  excreta. 


ISOLATION   OF  THE   ORGANISM  429 

Any  of  the  methods  described  for  the  isolation  of  the  typhoid  bacillus 
are  quite  applicable  to  the  isolation  of  the  paratyphoid  A  bacillus. 

Agglutination  reactions  with  the  patients'  serum  supplemented  by  absorp- 
tion tests  should  form  a  part  of  the  diagnosis  in  every  case. 

1.  The  most  important  step  is  to  demonstrate  the  presence  of  the  bacillus 
in  the  blood  stream.     (The  isolation  of  the  paratyphoid  A  bacillus  is  com- 
paratively easy  during  the  first  4  or  5  days  of  the  pyrexia  :   the  chances  of  a 
successful  blood  culture  are  greatly  diminished  by  the  8th  day,  even  when 
the  usual  5   c.c.   of  blood  is  withdrawn — cf.  enteric    fever    (Grattan   and 
Wood).) 

Grattan  and  Wood  sow  the  blood  (5  c.c.)  on  sterilized  ox  bile  and  after  incubating 
for  24  hours  at  37°  C.  plate  the  growth  on  Conradi-Drigalski's  original  medium, 
which  they  consider  better  than  the  more  recent  selective  media  l  ;  they  incubate 
again  and  then  pick  off  colonies  which  resemble  those  of  the  typhoid  bacillus,  which 
is  indistinguishable  on  the  Conradi-Drigalski  medium  from  the  paratyphoid  A 
bacillus. 

The  organism  isolated  must  be  fully  identified.  Firstly,  it  must  be  shown 
to  belong  to  the  paratyphoid  group  by  a  careful  study  of  its  morphological, 
staining,  cultural  and  fermentation  reactions.  (The  characteristics  of  the  para- 
typhoid A  bacillus  may  be  conveniently  summarized  here  :  it  is  a  short 
stumpy  gram-negative  bacillus  which  does  not  liquefy  gelatin,  forms  no 
indol,  does  not  clot  milk  but  turns  the  medium  permanently  acid,  ferments 
glucose,  mannite  and  dulcite  with  production  of  acid  and  gas,  but  has  no 
action  on  lactose  and  cane  sugar.)  Then  the  bacillus  must  be  examined,  as 
regards  its  agglutination  reactions,  with  known  typhoid,  paratyphoid  A  and 
paratyphoid  B  serums. 

In  the  case  of  an  organism  isolated  from  the  blood  these  tests  are  sufficient 
for  identification  (Grattan  and  Wood). 

2.  To  isolate  the  organism  from  the  urine  or  from  the  excreta  some  of  the 
material  should  be  sown  in  dulcite  broth  or  dulcite  peptone  water.     Dulcite 
is  the  enrichment  medium  par  excellence  for  the  paratyphoid  group  (Boycott). 
After  incubation — 2  or  3  days  sometimes  elapse  before  the  paratyphoid  A 
bacillus  produces  gas  in  dulcite  media,  but  ultimately  the  amount  formed  is 
considerable — some  of  the  culture  may  be  plated  on  Conradi-Drigalski's  or 
M'Conkey's    medium   and   the    organisms   isolated   tested   as   above.     But 
according  to  Grattan  and  Wood  when  a  bacillus  resembling  the  paratyphoid 
A  bacillus  is  isolated  from  the  stools  it  is  necessary  to  carry  the  identification 
a  step  further  (by  means  of  absorption  tests)  than  in  the  case  of  a  similar 
organism  isolated  from  the  blood. 

A  24-hour  growth  of  the  suspected  organism  on  agar  is  emulsified  in 
about  0'2  c.c.  of  paratyphoid  A  serum — which  is  diluted  according  to  its 
titre,  the  object  being  to  have  an  excess  of  bacilli  for  the  amount  of  agglutinin 
present :  e.g.  a  serum  having  a  titre  of  1-300  may  be  diluted  ten  times. 

Incubate  the  emulsion  for  2  hours  at  37°  C.,  centrifuge  and  put  up  the 
clear  supernatant  liquid  in  a  series  of  dilutions  in  sedimentation  tubes  and 
test  its  agglutinating  action  with  a  known  paratyphoid  A  bacillus. 

If  the  organism  used  for  absorption  be  the  paratyphoid  A  bacillus  then 
it  will  have  completely  removed  the  specific  paratyphoid  A  agglutinins  from 

1  None  of  these  media  will  differentiate  with  any  degree  of  certainty  the  typhoid  bacillus 
from  the  paratyphoid  bacilli.  That  however  is  immaterial  since  it  is  unlikely  that  they 
will  be  present  together.  The  main  use  of  these  media  is  to  differentiate  at  sight  the 
typhoid  and  paratyphoid  bacilli  from  the  colon  bacillus  which  is  invariably  present  and 
usually  in  large  numbers. 


430  THE  PARATYPHOID  A.   BACILLUS 

the  serum.  Grattan  and  Wood  say : — "  before  accepting  a  suspected 
organism  we  require  that  it  shall  completely  remove  the  agglutinin  specific 
for  the  paratyphoid  A  bacillus.  As  controls  we  have  frequently  tested 
heterologous  organisms  such  as  the  typhoid,  paratyphoid  B  and  colon  bacilli 
against  our  paratyphoid  A  serum  but  have  never  removed  the  agglutinin 
specific  for  the  paratyphoid  A  bacillus." 

3.  The  reactions  of  the  serum  of  patients  suffering  from  paratyphoid  A 
fever  have  been  described  above. 

The  pseudo-paratyphoid  A  bacillus. 

This  organism  appears  to  be  a  common  inhabitant  of  the  intestines  of  pigs  (Mor- 
gan) and  has  once  been  isolated  from  the  human  subject  during  some  investigations 
on  the  cause  of  summer  diarrhoea  (Morgan). 

The  pseudo-paratyphoid  A  bacillus  is  culturally  identical  with  the  paratyphoid 
A  bacillus  but  differs  from  the  latter  in  that  it  is  not  agglutinated  by  a  specific 
paratyphoid  A  serum. 


CHAPTER  XXVII.1 
THE  SALMONELLA  GKOUP. 

1.  Bacillus  paratyphosus  B. 
Introduction. 

Section  I. — Experimental  inoculation,  p.  433. 

Section  II. — Morphology,  p.  433. 

Section  III. — Biological  properties,  p.  434. 

Section  IV. — The  isolation  and  identification  of  the  bacillus,  p.  437. 

2.  Bacillus  enteritidis  Aertrycke. 
Introduction,  p.  438. 

Section  I. — Experimental  infection,  p.  439. 

Section  II. — Morphology,  p.  440. 

Section  III. — Biological  properties,  p.  440. 

Section  IV. — Isolation  and  identification  of  the  bacillus,  p.  441. 

3.  Bacillus  enteritidis  Gaertner. 
Introduction,  p.  442. 

Section  I. — Experimental  inoculation,  p.  442. 

Section  II. — Morphology,  p.  443. 

Section  III. — Biological  properties,  p.  443. 

Section  IV. — Isolation  and  identification  of  the  bacillus,  p.  444. 

4.  Pseudo-gaertner  bacilli,  p.  444. 

5.  Bacillus  typhi  murium,  p.  444. 

6.  Danysz's  virus,  p.  444. 

7.  The  bacillus  of  psittacosis,  p.  445. 
8    Bacillus  icteroides,  p.  445. 

1.    BACILLUS  PARATYPHOSUS  B. 

LIKE  the  paratyphoid  A  bacillus  the  paratyphoid  B  bacillus  was  first 
isolated  by  Schottmuller  in  1900-1  from  cases  of  disease  in  man  clinically 
indistinguishable  from  enteric  fever. 

In  England  the  description  "  paratyphoid  B  bacillus  "  is  limited  to  those 
strains  which  in  their  cultural,  agglutination  and  absorption  characteristics 
are  identical  with  the  strain  originally  isolated  by  Schottmuller  (p.  420). 

The  relationship  of  this  bacillus  to  the  other  organisms  of  the  Salmonella  group  has 
already  been  discussed  (p.  422)  and  the  position  may  be  summarized  by  stating  that 
in  England  the  following  species — all  identical  culturally — are  distinguished,  viz.  : — 

1.  Bacillus  paratyphosus  B.  (Schottmuller). 

1  This  chapter  has  been  rewritten. 


432  THE  SALMONELLA  GROUP 

2.  Bacillus  enteritidis  Aertrycke  (Durham,  De  Nobele)  vel  B.  suipestifer,  vel  B. 
cholerce  suis  (Salmon)  vel  bacillus  of  hog  cholera. 

3.  Bacillus  enteritidis  Gaertner. 

This  classification  is  unfortunately  not  adopted  on  the  Continent.  German 
observers,  as  Bainbridge  points  out,  regard  the  paratyphoid  B  bacillus  (Schottmiiller) 
and  the  aertrycke  bacillus  as  identical  species,  the  value  of  absorption  tests  not  having 
yet  been  acknowledged,  and  hence  considerable  confusion  results  from  reading  their 
observations.  The  two  names  are  retained  in  German  publications  but  merely  as 
labels  to  indicate  the  source  of  the  strain  :  if  obtained  from  a  human  source  it  is 
designated  the  paratyphoid  B  bacillus,  if  from  animals,  the  hog  cholera  bacillus. 
Consequently  what  in  English  nomenclature  is  a  paratyphoid  B.  bacillus  may  in 
German  be  an  aertrycke  bacillus,  and  conversely.  In  France  the  recent  important 
work  of  Bainbridge  and  O'Brien  has  not  yet  appeared  in  print. 

In  Europe  the  paratyphoid  B  bacillus  has  been  isolated  from  and  accepted  to  be 
the  cause  of  two  very  different  clinical  types  of  disease,  one  indistinguishable  by 
its  symptoms  from  enteric  fever,  the  other  characterized  by  the  symptoms  of  what 
is  generally  known  as  "  food-poisoning."  Whichever  type  the  symptoms  assume, 
the  disease  is  a  septiceemic  condition,  and  the  organism  can  be  recovered  from  the 
blood  during  life  and  from  the  spleen  after  death. 

The  former  is  by  far  the  more  common  of  the  two  types  of  infection,  and  in  Europe 
and  America  a  very  large  number  of  cases  of  paratyphoid  fever  due  to  the  para- 
typhoid B  bacillus  have  been  recorded.  In  England  it  is  estimated  that  about 
3-6  per  cent,  of  all  cases  of  enteric  fever  are  due  to  infection  with  the  paratyphoid 
B  bacillus,  in  America  about  10  per  cent,  and  in  Germany  also  about  10  per  cent. 
(Boycott).  In  South  Africa  paratyphoid  B  fever  appears  to  be  a  common  disease 
(M'Naught). 

Thus  in  these  countries  paratyphoid  fever  is  a  paratyphoid  B  infection ;  but  in 
India  the  disease  seems  almost  without  exception  to  be  caused  by  the  paratyphoid 
A  bacillus  (p.  423). 

As  regards  symptoms  of  food- poisoning  due  to  paratyphoid  B  the  present  informa- 
tion is  scanty  and  unreliable.  In  only  one  instance  at  present  has  the  paratyphoid 
B  bacillus  been  proved  to  be  present  in  cases  of  acute  gastro-enteritis  and  that 
instance  is  the  outbreak  recorded  by  Bainbridge  and  Dudfield.1 

The  German  accounts  of  food-poisoning  due  to  the  paratyphoid  B  bacillus  cannot 
be  accepted,  because  in  Germany  no  distinction  is.  drawn  between  the  paratyphoid 
B.  and  aertrycke  bacilli ;  and  the  latter  is  shown  by  Bainbridge  to  be  the  common 
cause  of  food- poisoning  (p.  438).  (This  observer  finds  that  all  the  organisms  isolated 
from  clinical  cases  of  food-poisoning  in  England  and  Germany  which  he  has 
examined  are  strains  of  the  aertrycke  bacillus.) 

Distribution  of  the  bacillus  in  the  body. — The  paratyphoid  B  bacillus  is  present  in 
the  blood-stream,  in  the  internal  organs  and  in  the  intestinal  contents  of  infected 
persons.  Apart  from  symptoms  of  paratyphoid  fever  and  food-poisoning  it  has 
been  found  in  the  gallbladder  in  cases  of  disease  of  the  gall  bladder  arid  in  persons 
in  apparently  good  health — :"  carriers.'*  Carriers  in  connexion  with  paratyphoid 
B  infections  were  first  investigated  by  Lentz.  Bainbridge  abstracted  the  records 
available  and  found  that  in  the  majority  of  cases  (26  out  of  29)  they  were  of  the 
female  sex,  that  a  striking  percentage  (7  out  of  26)  had  symptoms  of  biliary  disorders, 
and  that  in  every  case  their  blood  agglutinated  the  bacillus.  Paratyphoid  B  carriers 
are  therefore  very  like  enteric  carriers,  and  they  may  originate  epidemics  of  para- 
typhoid B  infection  (Sacquepee  and  Bellott,  Bainbridge  and  Dudfield). 

Apart  from  cases  of  paratyphoid  fever  and  persons  who  become  "  carriers,"  the 
bacillus  is  very  rarely  if  ever  found  in  the  human  intestine  or  urine  (Bainbridge  and 
O'Brien).  No  paratyphoid  B  bacilli  were  found  by  Morgan  in  summer  diarrhrea, 
by  Williams,  Rundle  and  Murray  in  healthy  children,  by  Seiffert,  and  by  Sobernheim 
in  healthy  men,  by  Bainbridge  and  O'Brien  in  convalescents  from  enteric  fever,  or 
by  Savage  in  enteric  fever  patients  (caused  by  the  typhoid  bacillus)  and  healthy 
persons. 

1  Journa*  of  Hygiene,  xi.  p.  24. 


THE   PARATYPHOID   B.   BACILLUS  433 

In  Germany  on  the  other  hand  "  paratyphoid  bacilli  "  i.e. — paratyphoid  B  and 
aertrycke — have  been  isolated  from  the  blood,  stools  and  urine  of  healthy  persons 
(Conradi,  Prigge  and  Sachs-Miike,  Gaethgens) ;  they  have  been  found  in  enteric 
fever  patients,  convalescents  and  contacts,  and  have  been  isolated  also  from  persons 
suffering  from  other  diseases  (Conradi). 

Distribution  of  the  bacillus  outside  the  body. — Bainbridge  failed  to  detect  the 
paratyphoid  B.  bacillus  in  the  excreta  of  50  healthy  pigs.  In  Germany  the  organism 
is  said  to  have  been  isolated  from  sausages  (Hiibener,  Rommeler),  milk,  meat,  etc. 
(p.  438).  Bainbridge  however  has  examined  a  number  of  these  so-called  paratyphoid 
B  bacilli  and  finds  that  the  strains  submitted  to  him  fall  into  two  groups,  viz.  : 

1.  Bacilli  of  the  aertrycke  type  which  were  all  obtained  from  food  or  from  cases 
of  food- poisoning. 

2.  Bacilli  of  the  paratyphoid  B  type  which  were  all  derived  from  cases  of  para- 
typhoid fever  and  paratyphoid  carriers. 

Bainbridge  therefore  is  of  opinion  that  the  normal  habitat  of  the  paratyphoid 
B  bacillus  is  the  human  alimentary  canal  and  bile  passages,  and  that  its  distribution 
is  limited  to  those  situations. 


SECTION   I.— EXPERIMENTAL  INOCULATION. 

The  pathogenicity  of  strains  of  the  paratyphoid  B.  bacillus  is  typically 
of  a  high  order  (Boycott).  Using  guinea-pigs  weighing  250  grams  and  broth 
cultures  incubated  at  37°  C.  for  20  hours  Boycott  found  that  the  inocula- 
tion of  1  c.c.  beneath  the  skin  led  to  death  in  18-40  hours,  and  that  O'l  c.c. 
intra-peritoneally  was  followed  by  death  in  less  than  18  hours. 

Feeding  experiments  were  negative  (Boycott). 


SECTION  II.— MORPHOLOGY. 

1.  Microscopical    appearance.    Staining    reactions. — The    paratyphoid    B 
bacillus  is  indistinguishable  as  regards  its  appearance  under  the  microscope 
and  in  its  staining  reactions  from  the  typhoid  and  paratyphoid  A  bacilli. 

It  is  a  stout,  motile  cocco-bacillus  tending  to  stain  more  deeply  at  the  ends 
than  in  the  centre  ;  in  culture  especially  on  gelatin  it  sometimes  grows  out 
into  filamentous  forms  ;  it  stains  with  ordinary  aniline  dyes  and  is  gram- 
negative. 

2.  Cultural  characteristics. — In  its  cultural  characteristics  the  paratyphoid 
B  bacillus  approaches  the  colon  bacillus. 

In  broth  it  grows  abundantly,  often  forming  a  pellicle  on  the  surface  and 
occasionally  giving  rise  to  a  faecal  odour. 

On  gelatin,  isolated  colonies  are  transparent  at  first  but  fairly  rapidly 
become  opaque  ;  occasionally  they  retain  the  "  iceberg  "  appearance.  In 
stroke  culture  the  bacillus  most  frequently  gives  rise  to  a  thick  whitish  layer 
which  becomes  opaque  and  viscous  as  the  culture  ages. 

On  potato  the  growth  generally  resembles  that  of  the  colon  bacillus — a 
thick,  brown  viscous  layer ;  a  glazed  appearance  is  uncommon. 

On  artichoke  the  culture  is  green  in  2  or  3  days. 

Milk. — Milk  is  not  coagulated  but  becomes  clear  and  about  the  second  week 
acquires  a  brownish  tint. 

Litmus  milk. — First  of  all  the  medium  becomes  slightly  acid,  the  litmus 
turning  red  :  but  after  about  3-7  days  a  secondary  alkalinity  develops,  the 
colour  of  the  litmus  reverting  to  blue. 

Litmus  whey. — In  the  first  few  days  a  small  amount  of  acid  is  formed 
turning  the  litmus  red,  afterwards  the  blue  colour  reappears. 

2E 


434 


THE  SALMONELLA  GROUP 


Vaccinated  media. — A  normal  growth  takes  place  on  media  which  have 
served  for  the  growth  of  the  typhoid  and  paratyphoid  A  bacilli.  On  the 
other  hand  on  media  which  have  served  for  the  growth  of  the  colon  bacillus 
the  culture  is  poor  ;  and  on  media  on  which  the  paratyphoid  B  bacillus  has 
been  grown  it  is  very  inconstant. 

Cultural  differences  between  the  paratyphoid  A.  bacillus  and  the 
paratyphoid  B.  bacillus. 


Cultures  on  gelatin. 
Cultures  in  broth. 


PARATYPHOID  A. 

Scanty   and   transparent 
(like  typhoid). 

Cloudy ;  no  pellicle. 


PARATYPHOID  B. 

Thick  and  opaque  (like 
the  colon  bacillus). 

Cloudy ;  pellicle  very 
common.  Often  fsecal 
odour. 


Cultures  on  potato. 

Faint     glaze     (like     the 
typhoid  bacillus). 

Thick,  pigmented  growth 
(like  the  colon  bacillus). 

Milk. 

No  coagulation. 

No  coagulation,  medium 
slowly  cleared. 

Litmus  milk. 

Permanently  red. 

Turned  red  first,  then 
blue. 

Litmus  whey. 

Permanently  red. 

First  red,  then  blue. 

SECTION  III.— BIOLOGICAL   PROPERTIES. 
1.  Biochemical  reactions. 

(a)  Action  on  carbohydrates. — The  paratyphoid  B.  bacillus  forms  acid 
and  gas  when  sown  in  glucose,  mannite,  dulcite,  Isevulose,  galactose,  arabinose, 
maltose  and  sorbite,  but  gives  rise  to  neither  -acid  nor  gas  in  lactose,  saccharose, 
salicin,  raffinose,  and  inulin. 

(/?)  Indol  production. — The  paratyphoid  B.  bacillus  produces  no  indol 
even  when  incubated  in  peptone  water  for  10  days. 

(7)  Neutral  red  media. — Very  often  the  colour  of  the  medium  is  reduced 
but  the  change  is  not  constant  (vide  paratyphoid  A). 

2.  Toxins. 

Cultures  of  paratyphoid  B.  contain  soluble  toxins.  A  dose  of  5-9  c.c. 
of  a  nitrate  of  a  culture  on  Martin's  broth  (2-8  days  at  37°  C.)  is  fatal  to 
guinea-pigs  when  inoculated  sub-cutaneously.  Franchetti  prepared  a  toxin 
which  was  fatal  to  rabbits  in  doses  of  1  c.c.  per  kg.  of  body  weight  when 
inoculated  intra-venously,  by  making  an  emulsion  of  cultures  in  sterile 
distilled  water,  shaking  for  2  days,  centrifuging  and  heating  to  44°  C.  for 
3  hours. 

Cathcart  grew  the  paratyphoid  B.  bacillus  on  agar  in  Roux  bottles,  washed 
off  the  growth  with  distilled  water  or  normal  saline  solution,  added  a  little 
toluol  and  after  repeatedly  shaking  for  8-10  days  and  then  heating  to  60°  C. 
for  half  an  hour  obtained  an  endotoxin  which  proved  fatal  to  mice  in  doses 
of  0*1  c.c.  intra-peritoneally  in  24  hours.  This  toxin  is  thermostable  at 
100°  C. 


THE   PARATYPHOID   B.   BACILLUS  435 


3.  Vaccination.    Properties  of  immune  serums. 

Animals  can  be  easily  immunized  by  inoculating  them  with  heated  or 
attenuated  cultures  (vide  infra).  Franchetti  has  prepared  an  agglu- 
tinating and  antitoxic  serum  by  inoculating  rabbits  with  his  toxin  (vide 
supra). 

Boycott  found  that  vaccination  with  the  paratyphoid  B.  bacillus  was 
protective  against  the  paratyphoid  B.  bacillus  but  not  against  the  aertrycke 
bacillus. 

4.  Agglutination. 

Agglutinins  are  developed  in  the  blood  in  response  to  a  naturally  acquired 
infection  in  man  and  to  the  inoculation  of  the  bacillus  in  animals. 

Serum  reactions  in  human  paratyphoid  B.  fever. — It  would  appear  that  a 
careful  determination  of  the  ultimate  limits  of  agglutination  will,  at  any 
rate  in  the  great  majority  of  cases,  give  accurate  information  as  to  the  nature 
of  a  typhoid  or  paratyphoid  [B.]  serum  (Boycott). 

As  a  rule  a  paratyphoid  B.  serum  has  little  agglutinating  action  on  the 
typhoid  bacillus  :  thus  in  a  case  recorded  by  Sacquepee  and  Rieux  a  para- 
typhoid B  serum  agglutinated  the  paratyphoid  B  bacillus  isolated  from  the 
patient  in  a  dilution  of  1-2000  and  the  typhoid  bacillus  in  only  1-40. 

But  anomalous  results  are  occasionally  observed.  Zupnik  has  shown  that 
the  serum  from  cases  of  enteric  fever — determined  by  isolation  of  the  typhoid 
bacillus — may  agglutinate  the  paratyphoid  B.  bacillus  in  as  high  or  even  in 
an  higher  dilution  than  it  agglutinates  the  homologous  organism.  Pratt 
found  that  the  serum  of  a  patient  from  whose  blood  he  isolated  the  typhoid 
bacillus  agglutinated  the  paratyphoid  B.  bacillus  in  a  dilution  of  1  in  200  and 
had  no  action  on  the  typhoid  bacillus  in  a  dilution  of  1  in  10. 

Normal  human  serum  may  agglutinate  the  paratyphoid  B.  bacillus  up  to 
a  dilution  of  1  in  100.  But  in  paratyphoid  B.  fever  the  serum  reaction  is 
usually  manifested  in  relatively  high  dilutions — up  to  1-1000  ;  and  extra- 
ordinarily active  human  serums  have  been  encountered  by  Savage  (1-70,000) 
and  Zupnik  (1-140,000).  Boycott's  three  cases  all  agglutinated  in  a  dilu- 
tion of  1-5000,  and  in  only  one  of  21  cases  observed  by  Zupnik  was  the 
reaction  less  than  1-1000. 

Paratyphoid  B  fever  cannot  be  diagnosed  on  a  demonstration  that  the 
serum  of  a  person  suffering  from  an  illness  resembling  enteric  fever  fails  to 
agglutinate  the  typhoid  bacillus  even  in  low  dilutions.  And  still  less  is  a 
diagnosis  of  paratyphoid  fever  justified  by  the  bare  fact  that  agglutination 
is  observed  with  a  paratyphoid  B  or  similar  bacillus  (Boycott).  Forty-one 
per  cent,  of  typhoid  serums  (in  a  consecutive  series  of  86)  agglutinated  the 
paratyphoid  B  bacillus  (Boycott). 

In  the  paratyphoid  B  food-poisoning  epidemic  investigated  by  Bainbridge 
and  Dudfield  the  serum  of  the  persons  involved  agglutinated  the  para- 
typhoid B  bacillus  in  dilutions  of  1-100  to  1-400. 

Animal  serums. — The  serum  of  animals  inoculated  with  the  paratyphoid  B 
bacillus  acquires  the  property  of  agglutinating  the  bacillus  in  high  dilution. 
At  the  same  time  co-agglutinins  are  developed,  especially  for  the  aertrycke 
bacillus  ;  indeed  the  amount  of  co-agglutinin  for  this  organism  may  be  so 
considerable  as  to  be  almost  equal  in  amount  to  the  specific  agglutinin. 

Bainbridge  finds  that  if  immunization  be  effected  with  living  bacilli,  instead 
of  with  sterilized  cultures,  the  amount  of  co-agglutinin  is  much  less  and  the 
titre  of  the  specific  agglutinin  higher. 

In  the  case  of  the  paratyphoid  B  bacillus,  which  is  relatively  an  highly 


436  THE  SALMONELLA  GROUP 

pathogenic  organism,  immunization  is  most  successfully  carried  out  by 
inoculating  the  animal  thus 

1st  day,        -        O'OOl  c.c.  of  a  20-hour  broth  culture  intra-venously 

15th  day,      -        O'Ol  c.c. 

29th  day,      -         O'Ol  c.c.  „  „  „ 

and  bleeding  it  on  the  8th  day  after  the  last  injection  from  the  carotid  after 
ansesthetization.  In  this  way  it  is  easy  to  obtain  a  serum  with  a  specific 
titre  of  1-20,000,  and  even  of  1-40,000. 

An  anti-paratyphoid  B  serum  has  little  action  on  the  typhoid,  paratyphoid 
A  and  gaertner  bacilli,  but  agglutinates  the  aertrycke  bacillus  often  in  high 
dilution,  hence  the  confusion  which  has  until  recently  existed  between  that 
organism  and  the  paratyphoid  B  bacillus.  And  the  large  amount  of  aertrycke 
co-agglutinin  in  a  paratyphoid  B  serum,  and  paratyphoid  B  co-agglutinin  in 
an  aertrycke  serum,  is  obviously  the  explanation  of  the  "  irregularity  "  of  the 
action  of  a  "  paratyphoid  B  "  serum  on  a  "  paratyphoid  B  "  bacillus. 

5.  Absorption  tests. 

By  means  of  absorption  tests  the  true  nature  of  a  serum  or  of  an  unknown 
bacillus  of  the  typhoid,  colon,  or  paratyphoid  groups  is  readily  determined. 

In  the  case  of  a  paratyphoid  B.  serum  the  whole  of  the  specific  agglutinin 
as  well  as  the  co-agglutinins  are  removed  by  saturating  with  a  paratyphoid  B. 
bacillus  :  whereas  by  saturating  with  for  example  the  aertrycke  bacillus  only 
the  co-agglutinin  for  that  organism  is  removed,  leaving  the  specific  agglutinin 
intact. 

Similarly,  in  the  case  of  an  aertrycke  serum  all  the  specific  agglutinin  and 
the  co-agglutinins  are  removed  by  saturating  with  an  aertrycke  bacillus, 
but  by  saturating  with  a  paratyphoid  B.  bacillus  only  the  co-agglutinin  for 
the  paratyphoid  B.  bacillus  is  removed. 

It  is  due  to  these  observations  that  the  paratyphoid  B.  bacillus  is  dis- 
tinguished from  the  aertrycke  bacillus,  and  recognized  to  be  an  independent 
species. 

Technique. 

Assume  that  a  paratyphoid  B  serum  has  an  agglutination  titre  of  20,000. 

1.  Take  O'l  c.c.  of  the  serum  and  add  4'9  c.c.  of  normal  saline  solution.     This 
will  give  5  c.c.  of  a  1  in  50  dilution  of  the  serum.     Divide  into  two  equal  portions  of 
2-5  c.c.  each. 

2.  Take  five  large  agar  slope  cultures  (24  hours)  of  the  paratyphoid  B  bacillus, 
scrape  off  the  growth  with  a  platinum  wire  and  add  it  to  one  portion  of  the  diluted 
serum.     (The  growth  should  not  be  washed  off  with  the  diluted  serum.) 

3.  Emulsify  similarly  in  the  second  portion  of  the  diluted  serum  five  large  agar 
slope  cultures  of  the  aertrycke  bacillus. 

4.  Incubate  both  emulsions  at  37°  C.  for  2  hours. 

5.  Centrifuge  the  emulsions  until  the  whole  of  the  bacilli  are  precipitated  and  the 
supernatant  fluids  are  clear. 

6.  Pipette  off  the  supernatant  fluids  into  separate  sterile  tubes. 

7.  Test  the  agglutinating  reactions  of  both  fluids  up  to  a  dilution  of  1—20,000 
against  both  the  paratyphoid  B  bacillus  and  the  aertrycke  bacillus,  remembering 
that  the  serum  is  already  diluted  1  in  50. 

Before  absorption  the.  serum  agglutinated  both  the  paratyphoid  B.  bacillus  and 
the  aertrycke  bacillus  in  high  dilution  (1-20,000  and  1-10,000).  After  absorption 
with  the  paratyphoid  B  bacillus  the  titre  for  both  organisms  was  reduced  to  about 
1-100  to  1-400.  After  absorption  with  the  aertrycke  bacillus  the  titre  for  that 
organism  had  fallen  to  about  1-100  to  1-400  while  the  titre  for  the  paratyphoid  B 
bacillus  was  unchanged  or  only  slightly  reduced. 

The  amount  of  bacilli  to  be  added  must  necessarily  vary  according  to  the 
titre ;  the  higher  the  titre  the  more  bacilli. 


THE   PARATYPHOID   B.   BACILLUS 


437 


The  following  table  taken  from  Bainbridge  and  O'Brien's  paper  will  make 
the  above  statements  clear. 


SERUM. 

AGGLUTINATION  LIMITS  FOR 

Bacillus  F. 

B.  aertrycke. 

B.  Paratyphoid  B. 

1.  Bacillus  F  1  (original  titre), 

5,000 

5,000 

2,000 

Absorbed  with  bacillus  F, 

<200 

<200 

<200 

„             „    B.  aertrycke,  - 

<200 

<200 

<200 

„    B.  paratyph.  B, 

5,000 

5,000 

<200 

2.  Bacillus     aertrycke    (original 

titre), 

10,000 

10,000 

5,000 

Absorbed  with  bacillus  para- 

typh. B      - 

10,000 

10,000 

<200 

Absorbed  with  B.  F,       - 

<50 

<50 

<50 

3.  B.    paratyphosus   B  (original 

titre), 

5,000 

10,000 

20,000 

Absorbed   with    bacillus    aer- 

trycke,        .... 

<400 

<400 

10,000 

Absorbed  with  B.  F, 

<400 

<400 

10,000 

6.  Complement  fixation. 

By  means  of  the  complement  fixation  reaction  H.  R.  Dean  has  shown  that 
the  paratyphoid  B  bacillus  can  be  clearly  differentiated  from  the  typhoid 
and  paratyphoid  A  bacilli  and  also  from  the  aertrycke  bacillus  (p.  428  for 
technique). 

Unless  a  suitable  dilution  of  antiserum  be  employed  the  group  antibodies 
in  the  serum  will  mask  the  specificity  of  the  reaction.  Dean  has  however 
shown  that  these  group  antibodies  can  be  removed  by  absorption  previously 
to  carrying  out  the  complement-fixation  reaction.  Thus,  when  a  para- 
typhoid B  antiserum  is  absorbed  with  an  emulsion  of  paratyphoid  A  bacilli, 
the  serum  loses  its  capacity  for  binding  complement  in  the  presence  of  either 
a  paratyphoid  A  extract  or  a  typhoid  extract.  After  absorption  with  a  para- 
typhoid B  emulsion  the  group  antibodies  are  found  to  be  removed,  together 
with  the  antibodies  specific  for  paratyphoid  B. 


SECTION  IV.— ISOLATION   AND   IDENTIFICATION   OF  THE 
PARATYPHOID   B.   BACILLUS. 

In  cases  of  paratyphoid  fever  the  organism  may  be  isolated  from  the 
blood-stream,  or  from  the  stools  or  urine,  and  after  death  from  the  spleen. 

In  localized  paratyphoid  infections— e.g.  cholecystitis,  gall  stones,  abscesses 
in  various  parts  of  the  body— material  (bile,  pus,  etc.),  from  the  site  of  infec- 
tion must  be  used. 

The  material  should  be  sown  in  dulcite  (1-2  per  cent.)  broth  or  dulcite 
peptone  water  incubated  at  37°  C.  for  24  hours  or  longer — in  some  cases  the 


1  Bacillus  F  was  an  organism  isolated  by  Williams.  Rundle  and  Murray  from  cases 
of  summer  diarrhoea  and  shown  by  this  test  to  be  identical  with  B.  aertrycke. 


438  THE   SALMONELLA  GROUP 

fermentation  of  dulcite  is  delayed — and  then  a  loopful  plated  out  on  Conradi- 
Drigalski's  or  MacConkey's  medium. 

After  incubating  the  plates  for  24  hours  a  number  of  the  colourless  colonies 
are  picked  off  and  tested  as  regards  their  morphology,  staining  reactions, 
cultural  characteristics  (gelatin  slopes  and  litmus  milk)  and  biological  pro- 
perties (glucose,  lactose). 

If  bacilli  be  isolated  having  up  to  this  point  all  the  characteristics  of  the 
paratyphoid  B  bacillus  it  is  absolutely  necessary,  in  order  to  definitely  identify 
the  organism,  to  test  its  reaction  with  a  known  serum  both  before  and  after 
absorption.  If  a  specific  serum  be  not  available  it  will  be  necessary  to 
inoculate  a  rabbit  with  minute  doses  of  a  living  culture  intra-venously 
(vide  supra)  and  to  test  the  action  of  this  serum  on  a  known  paratyphoid  B 
and  a  known  aertrycke  bacillus  both  before  and  after  absorption. 


2.    BACILLUS  ENTERITIDIS  AERTRYCKE. 

Synonyms  :  Bacillus  of  hog  cholera  :   Bacillus  suipestifer  :   B.  cholerse  suis. 

The  aertrycke  bacillus  was  isolated  in  1898  from  epidemics  of  food- 
poisoning  by  Durham  in  England  and  De  Nobele  in  Belgium  and  has 
become  known  as  the  aertrycke  bacillus  from  the  name  of  the  village  in 
which  De  Nobele's  epidemic  occurred. 

A  bacillus  known  as  the  hog-cholera  bacillus  or  bacillus  suipestifer  had 
previously  (in  1885)  been  isolated  by  Salmon  and  Theobald  Smith  from  swine 
suffering  from  a  disease  known  as  swine  fever  or  hog  cholera  (Ger.  Schweine- 
pest). 

These  two  organisms  are  now  admitted  to  be  identical. 

There  is  at  present  no  uniformity  in  the  nomenclature  of  the  bacillus,  some  writers 
referring  to  it  as  the  Bacillus  suipestifer  or  bacillus  of  hog  cholera,  others  as  the 
aertrycke  bacillus.  Bacillus  suipestifer  is  undoubtedly  its  original  name  but  the 
term  is  misleading,  as  it  implies  a  relationship  which  has  been  proved  not  to  exist :  1 
hence  it  has  seemed  better  to  adopt  the  title  Bacillus  enteritidis  Aertrycke  which 
represents  the  usual  role  of  the  organism  in  human  pathology. 

Occurrence  in  man. — The  aertrycke  bacillus  appears  to  be  the  organism  most 
frequently  found  in  cases  of  "  food  poisoning  "  in  man.  "  Food  poisoning  "  is  an 
acute  septicsemic  condition  accompanied  by  vomiting,  diarrhoea  and  collapse,  and 
in  severe  cases  terminating  fatally. 

In  healthy  human  subjects  the  organism  does  not  appear  to  be  present. 

Morgan  isolated  2  strains  of  the  bacillus  from  among  303  cases  of  summer  diarrhoea, 
and  Williams,  Bundle,  and  Murray  isolated  a  bacillus — Bacillus  F — now  admitted 
to  be  identical  with  the  aertrycke  bacillus  (p.  437)  from  cases  of  the  same  disease 

In  the  lower  animals  the  organism  appears  to  have  a  wider  distribution.  Petrie 
and  O'Brien  found  it  frequently  in  healthy  guinea-pigs,  and  O'Brien  has  described 
several  guinea-pig  carriers. 

In  Germany  "paratyphoid"  bacilli — probably  the  aertrycke  bacillus  (Bain- 
bridge) — have  been  isolated  from  the  intestines  of  healthy  pigs  (Uhlenhuth  8  per 
cent.,  Seiffert  3 '5  per  cent.),  from  milk  (Fischer),  water  (Conradi),  and  sausages 
(Hiibener,  Rommeler).  Bainbridge  and  also  Savage  have  failed  to  detect  the 
presence  of  this  organism  in  the  intestines  or  meat  of  healthy  animals  in  England. 

x  It  should  perhaps  be  pointed  out  here  that  in  the  opinion  of  Lourens  and  Glasser  the 
view  that  hog  cholera  is  due  to  a  "  filter-passer  "  is  not  borne  out  by  their  own  observa- 
tions. According  to  these  experimenters  hog  cholera  is  due  to  Salmon's  bacillus  which 
can  disintegrate  into  particles  small  enough  under  certain  circumstances  to  pass  through 
porcelain  niters ;  that  in  all  cases  occurring  during  an  epidemic  the  bacillus  is  found 
with  the  filter-passer ;  and  it  is  certain  that  the  bacillus  alone  can  produce  the  disease. 
This  view  is  contested  by  Uhlenhuth. 


THE   AERTRYCKE   BACILLUS  439 

Bainbridge  and  O'Brien  are  of  opinion  that  the  normal  habitat  of  the  organism 
is  the  alimentary  canal  of  pigs  and  perhaps  of  other  domestic  animals,  and  the 
meat  derived  from  such  animals,  but  that  in  England  such  occurrence  is  rare. 

In  many  epidemics  among  pigs  suffering  from  hog  cholera  the  bacillus  is  present 
in  small  numbers  in  the  blood  and  in  enormous  numbers  in  the  intestinal  lesions,  in 
the  juice  of  the  lungs,  in  the  bronchial  mucus,  urine,  lymphatic  glands,  liver,  spleen 
and  excreta.  It  is  not  infrequently  accompanied  in  these  cases  by  other  micro- 
organisms of  secondary  infection  such  as  the  colon  bacillus.  Swine  become  infected 
by  eating  food  contaminated  with  the  excreta  of  infected  animals. 

SECTION  I.— EXPERIMENTAL  INFECTION. 

Guinea-pigs,  rabbits,  rats  and  mice  are  very  susceptible  to  inoculation 
with  the  aertrycke  bacillus.1  These  animals  may  be  infected  by  inoculating 
them  either  beneath  the  skin,  in  the  peritoneal  cavity  or  in  the  muscles. 

Guinea-pigs. — The  bacillus  is  extremely  pathogenic  to  guinea-pigs  (Petrie 
and  O'Brien).  Doses  of  O001  c.c.  of  a  young  broth  culture  invariably  caused 
the  death  of  guinea-pigs  weighing  250  grams  in  about  5  days  :  in  some  cases 
doses  of  O000,l  and  0'000,001  c.c.  proved  fatal.  Guinea-pigs  however  are 
not  readily  killed  by  feeding  them  with  cultures  of  the  bacillus. 

White  rats. — Doses  of  2  c.c.  of  a  young  broth  culture  were  fatal  in  6  days 
when  given  sub-cutaneously  and  1  c.c.  intra-peritoneally  was  fatal  in  24 
hours. 

Mice. — Whether  inoculated  beneath  the  skin  or  into  the  peritoneal  cavity 
a  dose  of  0*01  c.c.  of  a  young  broth  culture  is  fatal  within  2  days. 

Rabbits. — O'l  c.c.  of  a  young  broth  culture  sub-cutaneously  killed  a  rabbit 
weighing  900  grams  in  2  days,  O'l  c.c.  intra-peritoneally  was  fatal  to  a 
rabbit  weighing  1300  grams  in  24  hours,  and  0*01  c.c.  intra-venously  killed  a 
rabbit  weighing  1300  grams  in  5.  days  (Petrie  and  O'Brien). 

Post  mortem,  the  principal  feature  in  acute  cases  is  intense  local  haemor- 
rhagic  oedema,  and  in  more  chronic  cases  necrosis  or  abscess  at  the  site  of 
inoculation  :  it  is  rare  to  find  even  minute  spots  in  the  liver  and  spleen  in 
experimental  animals  (Petrie  and  O'Brien).2 

Pigeons  are  less  susceptible  to  infection  ;  a  fatal  result  can  be  obtained 
only  by  using  considerable  doses  and  inoculating  into  the  muscles  or  beneath 
the  skin.  They  cannot  be  infected  by  feeding. 

The  virulence  of  the  bacillus  can  be  increased  in  a  very  extraordinary 
manner  by  passing  it  through  a  series  of  pigeons.  Selander,  by  passing  a 
virulent  strain  which  had  already  been  passed  through  several  rabbits  through 
a  series  of  pigeons,  obtained  a  virus  which  was  so  highly  virulent  that  0*05  c.c. 
of  the  blood  of  the  pigeon  when  inoculated  into  the  ear  vein  of  a  normal 
rabbit  killed  the  animal  in  4  or  5  hours  :  the  bacilli  in  the  blood  of  this  pigeon 
were  thirty  times  more  numerous  than  the  red  cells. 

Swine. — Swine  are  only  slightly  susceptible  to  sub-cutaneous  inoculation 
of  cultures  of  standard  virulence,  and  to  overcome  this  insusceptibility  a 
virus  the  virulence  of  which  has  been  increased  by  passing  it  through  pigeons 
must  be  used.  Intra- venous  inoculation  is  more  severe  and  invariably 

1  A  recently  isolated  strain  must  be  used  ;  in  culture  the  organism  soon  loses  its 
pathogenicity. 

2  These  appearances  may  be  compared  with  those  found  in  two  epizootics  among 
guinea-pigs,  which  the  same  observers  attributed  to  a  filter-passing  organism  but  from 
many  of  the  animals  affected  with  which  the  aertrycke  bacillus  was  isolated.     The 
intestines  of  these  animals  were  often  congested ;   the  liver  and  spleen  were  usually  con- 
gested, and  occasionally  contained  small  grey  or  yellow  nodules  :    in  some  of  the  cases 
the  supra-renals  showed  varying  degrees  of  congestion,  and  patchy  congested  areas  were 
often  seen  in  the  lungs  :   effusions  into  the  serous  cavities  did  not  occur. 


440  THE   SALMONELLA  GROUP 

proves  fatal :  the  symptoms  are  the  same  as,  and  post  mortem  the  lesions  are 
identical  with,  those  found  in  the  spontaneous  disease.  A  like  result  follows 
if  swine  be  fed  with  food  which  has  been  mixed  with  a  large  quantity — up  to 
a  litre — of  a  young  culture  of  a  recently  isolated  strain. 

There  are,  however,  essential  differences  between  the  disease  produced  by  the 
inoculation  of  the  aertrycke  bacillus  (bacillus  suipestifer)  and  the  spontaneous  dis- 
ease of  hog  cholera.  In  the  latter  the  disease  is  transmissible  from  the  sick  to  the 
healthy,  the  blood  is  infectious,  and  if  the  animal  recover,  it  is  permanently  immune 
in  a  high  degree  :  in  the  former  all  these  characteristics  are  absent ;  the  explanation 
being  that  the  spontaneous  disease  is  caused  by  a  filtrable  virus  with  which  the 
bacillus  aertrycke  (suipestifer)  is  associated  merely  as  a  secondary  infection. 

Sheep,  cows  and  calves  can  only  be  infected  by  intra-venous  inoculation. 

SECTION  II.— MORPHOLOGY. 

1.  Microscopical  appearance. — The  bacillus  aertrycke  has  the  microscopical 
appearances  and  staining  reactions  common  to  the  members  of  the  typhoid- 
colon  group.     In  tissues  and  infected  fluids  the  bacillus  is  always  non-motile, 
but  in  broth,  agar  or  gelatin  cultures  it  is  highly  motile  and  has  four  to  seven 
flagella  (Ferrier). 

Metchnikoff  has  drawn  attention  to  the  marked  pleomorphism  of  the 
aertrycke  bacillus  and  has  pointed  out  that  in  cultures  long  filaments  as 
well  as  cocci  sometimes  arranged  in  chains  are  seen,  in  addition  to  the 
typical  cocco-bacillary  forms. 

2.  Cultural  characteristics. — The  cultural  characteristics  of  the  aertrycke 
bacillus  are  identical  in  every  respect  with  the  paratyphoid  B  bacillus  and 
with  the  gaertner  bacillus. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 

1.  Vitality  and  virulence. — The  aertrycke  bacillus  retains  its  vitality  and 
virulence  in  cultures  for  several  months. 

Cornil  and  Chantemesse  obtained  an  attenuated  bacillus  by  exposing  a 
culture  to  a  temperature  of  43°  C.  for  90  days.  The  virulence  of  such  an 
attenuated  strain  may  be  restored  to  normal  by  passing  it  through  a  series 
of  rabbits ;  moreover  the  virulence  of  a  normal  strain  may  be  raised  to  a 
very  high  degree  by  passage  through  a  series  of  pigeons. 

The  bacillus  is  killed  by  heating  it  at  54°  C.  for  40  minutes  (Selander).  In 
brine  it  appears  to  be  capable  of  living  for  a  considerable  length  of  time 
(Savage). 

2.  Biochemical    reactions,    (a)  Action    on    carbohydrates.— The    changes 
produced  by  the  aertrycke  bacillus  when  grown  in  media  containing  various 
carbohydrates  are  the  same  as  those  produced  by  the  paratyphoid  B  and 
gaertner  bacilli. 

(b)  Indol. — The  aertrycke  bacillus  produces  no  indol  in  peptone  water. 

3.  Vaccination. — Cornil  and  Chantemesse  vaccinated  rabbits  by  inoculating 
them  with  their  attenuated  cultures  :    MetchnikofE  immunized  rabbits  by 
inoculating  small  doses  of  the  virus. 

The  inoculation  of  swine  with  pure  cultures  of  the  bacillus  produces  no 
immunity  .(Dorset,  MacClintock),  or  merely  a  transitory  immunity  (Lourens) 
against  hog  cholera. 

4.  Toxins.     In  the  tissues  of  inoculated  animals  the  aertrycke  bacillus 
produces  a  very  powerful  toxin  (Selander). 

The  blood  of  rabbits  which  have  succumbed  to  inoculation  of  a  virus  of 


THE  AERTRYCKE   BACILLUS 


441 


increased  virulence,  after  being  heated  at  57°  C.  for  an  hour  to  destroy  any 
organisms  present  therein,  is  sufficiently  virulent  to  cause  the  death  of  normal 
rabbits  in  3-4  hours  when  inoculated  in  doses  of  4-8  c.c. 

The  toxin  appears  to  be  produced  in  only  minimal  quantity  in  cultures. 
MacFadyen  by  triturating  the  bacilli  in  liquid  air  (p.  379)  obtained  a  toxin 
which  killed  rabbits  when  inoculated  intra-venously  in  doses  of  1  c.c. 

5.  Agglutination. — The  serum  of  persons  suffering  from  "  food-poisoning  " 
due  to  the  aertrycke  bacillus  agglutinates  that  bacillus  but  has  little  action 
on  the  gaertner  bacillus. 

The  serum  of  animals  immunized  with  the  aertrycke  bacillus  agglu- 
tinates the  homologous  bacillus  and  contains  co-agglutinins  in  considerable 
amount  for  the  paratyphoid  B  bacillus,  but  has  little  or  no  action  on  the 
gaertner  bacillus. 

By  means  of  the  agglutination  reaction  with  the  serum  of  immunized 
animals  the  aertrycke  bacillus  is  therefore  at  once  distinguished  from  the 
gaertner  bacillus,  but  by  a  simple  determination  of  the  limits  of  agglutination 
it  is  generally  impossible  to  differentiate  it  from  the  paratyphoid  B  bacillus, 
both  organisms  being  agglutinated  by  the  respective  specific  serums  in  about 
the  same  titre  (p.  435).  To  distinguish  between  the  aertrycke  and  the  para- 
typhoid B  bacilli  it  is  consequently  necessary  to  adopt  the  method  of 
absorption  of  agglutinins  (p.  436)  (Boycott,  Bainbridge). 

6.  Absorption  tests. — If  the  agglutination  titre  of  an  antiaertrycke  serum 
be  determined  both  for  its  own  organism  and  also  for  the  paratyphoid  B 
bacillus  the  two  determinations  will  in  most  cases  be  found  to  be  approximately 
the  same.     On  absorbing  such  a  serum  with  its  homologous  bacillus  all  the 
homologous  agglutinins  as  well  as  the  heterologous  co-agglutinins  will  be 
removed.    'But  if  absorbed  with  its  heterologous  bacillus  the  heterologous 
co-agglutinins  will  be  almost  entirely  removed  while  the  serum  will  still 
retain  its  power  of  agglutinating  the  homologous  bacillus  in  high  dilution  ; 
differentiation  is  thus  rendered  possible.     The  following  table  taken  from 
Bainbridge  (Jr.  Path,  and  Bact.  xiii.  p.  453)  will  ftiake  these  points  clear. 


SERUM. 

AGGLUTINATION  LIMIT. 

B.  paratyphoid  B. 

B.  aertrycke. 

B.  aertrycke. 
Original  titre  :  — 

10,000 

10,000 

Absorbed  with  — 
B.  aertrycke  :  — 
B.  paratyphoid  B  :  — 

100 
<100 

100 
10,000 

7.  Complement  fixation. — By  using  an  homologous  bacillary  extract  and  a 
suitable  dilution  of- an  antiserum  the  aertrycke  bacillus  can  be  clearly  dif- 
ferentiated from  the  paratyphoid  B  bacillus  (Dean)  (p.  437). 


SECTION  IV.— ISOLATION  AND   IDENTIFICATION   OF  THE 
AERTRYCKE   BACILLUS. 

In  cases  of  food-poisoning  the  organism  should  be  looked  for  in  the  suspected 
meat,  in  the  blood  of  the  patient  and  in  the  stools  ;  and  should  a  case  prove 
fatal,  in  the  spleen  and  intestinal  contents. 


442  THE   SALMONELLA  GROUP 

The  method  will  be  the  same  as  for  the  isolation  of  the  paratyphoid  B 
bacillus,  viz. :  preliminary  enrichment  in  dulcite  peptone  water  with  sub- 
sequent plating  on  neutral-red-lactose  agar  or  Conradi-Drigalski's  medium. 
Suspicious  colonies  will  then  be  examined  as  regards  their  biological  pro- 
perties— fermentation  reactions  and  production  of  indol.  The  identification 
of  the  organism  must  finally  rest  upon  a  study  of  its  reaction  with  known 
serums.  The  impossibility  of  distinguishing  it  from  the  paratyphoid  B 
bacillus  except  by  an  investigation  of  absorption  tests  must  be  emphasized. 
If  specific  serums  be  not  available  the  bacillus  must  be  inoculated  into  a 
rabbit  by  the  method  described  at  p.  435,  and  the  serum  of  the  rabbit  used  for 
agglutination  and  absorption  tests. 

3.    BACILLUS  ENTERITIDIS  GAERTNER. 

The  gaertner  bacillus  was  isolated  by  Gaertner  at  Frankenhausen 
in  1888  from  some  meat  which  was  suspected  to  have  been  the  cause 
of  an  epidemic  of  "  food-poisoning  "  (p.  438)  and  also  from  the  spleen  of  the 
patient  who  died  as  a  result  of  the  infection. 

As  a  cause  of  "  food -poisoning  "  the  gaertner  bacillus  seems  to  be  less  common 
than  the  aertrycke  bacillus,  but  epidemics  attributed  to  it  have  been  recorded  by 
van  Ermengem  at  Morseele,  Brussels  and  Gand,  by  M'Weeney  at  Limerick  and  by 
other  observers  ;  while  Bain  bridge  mentions  eleven  epidemics  in  which  Gaertner'^ 
bacillus  was  identified.  In  most  of  the  cases  in  which  the  origin  of  the  incriminated 
food  was  traced,  it  was  found  to  have  been  derived  from  animals  which  were  ill  at 
the  time  of  slaughter. 

Apart  from  epidemics  of  food  poisoning  the  organism  has  rarely  been  recorded 
in  man — four  times  by  Morgan  in  cases  of  summer  diarrhoea  and  once  by  Savage  in 
a  case  of  enteric  fever. 

The  gaertner  bacillus  seems  to  cause  an  epizootic  disease  among  animals.  Such 
epizootics  have  been  recorded  in  rats  and  rabbits  (Boycott,  Dunbar,  Bainbridge) 
and  in  guinea-pigs  (Bainbridge  and  O'Brien). 

According  to  Uhlenhuth  Gaertner' s  bacillus  is  a  normal  inhabitant  of  the  rat's 
intestine. 

In  healthy  cattle  Gaertner's  bacillus  would  appear  to  be  of  very  uncommon 
occurrence  ;  Sobernheim  isolated  it  only  twice  from  a  very  large  number  of  such 
animals,  and  Savage,  Miiller  and  others  have  failed  to  detect  it  under  similar 
conditions. 

In  sick  cattle  the  organism  has  been  isolated  from  calves  suffering  from  diarrhoea, 
from  cows  in  cases  of  post  partum  sepsis,  and  from  young  calves  in  which  possibly 
infection  took  place  through  the  umbilical  cord. 

Savage  has  recently  recorded  finding  it  in  a  sausage. 

Boycott  has  described  a  spontaneous  methsemoglobinsemia  in  rats  due  to  infection 
with  the  gaertner  bacillus. 

The  relation  of  the  gaertner  bacillus  to  other  members  of  the  paratyphoid 
group  is  considered  in  Chap.  XXV. 

SECTION  I.— EXPERIMENTAL   INOCULATION. 

Recently  isolated  strains  of  Gaertner's  bacillus  are  highly  virulent  to  guinea- 
pigs,  rabbits  and  mice  on  sub-cutaneous  and  especially  on  intra-peritoneal 
inoculation  (^i^  mg.  of  a  moist  culture).  Post  mortem  examination  shows 
hypersemia  of  the  lungs,  spleen,  supra-renal  capsules,  etc.  :  in  many  cases 
small  areas  of  necrosis  are  seen  in  the  liver. 

To  infect  animals  by  feeding  them  with  cultures  of  the  bacillus  large 
quantities  of  material  have  to  be  used  :  the  young  of  a  species  are  generally 
more  susceptible  to  this  mode  of  infection  than  adults  of  the  same  species. 

Feeding  experiments. — Mice  fed  on  the  muscles,  spleen,  liver,  etc.  of  the 


THE   GAERTNER    BACILLUS 


443 


carcase  of  a  guinea-pig  killed  with  a  dose  of  living  virulent  culture  died  in 
8-15  days  when  the  meat  was  given  uncooked,  and  in  30-38  days  when  the 
meat  was  boiled  before  being  given  to  them  (Cathcart). 

SECTION  II.— MORPHOLOGY. 

In  its  microscopical  appearances  and  staining  reactions  the  gaertner  bacillus 
is  indistinguishable  from  the  other  members  of  the  typhoid-colon  group. 

On  the  ordinary  media  it  grows  readily  yielding  growths  which  are  in  no 
way  different  from  those  of  the  paratyphoid  B  bacillus  and  of  the  aertrycke 
bacillus. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 

1.  Biochemical  reactions,     (a)  Action  on  carbohydrates.— The  fermentation 
reactions  of  the  gaertner  bacillus  are  identical  with  those  of  the  paratyphoid 
B  and  aertrycke  bacilli. 

(/3)  Production  of  indol. — Gaertner's  bacillus  produces  no  indol  when 
grown  in  peptone  water. 

2.  Toxin.— The  gaertner  bacillus  contains  a  toxin  of  the  endotoxin  type 
(Cathcart).     The  most  powerful  toxin  is  obtained  by  washing  off  the  bacilli 
from  an  agar  culture  in  a  Roux  bottle  with  distilled  water  or  normal  saline 
solution,   adding  a  little  toluol,   allowing  to  autolyze  for  8-10  days — the 
bottles  being  repeatedly  shaken  during  this  process — and  then  sterilizing  by 
heat  (60°-100°C.  for  30  minutes).     Heated  extracts  are  more  toxic  than 
unheated,  and  the  toxin  produced  in  this  manner  will  withstand  a  tempera- 
ture of  100°  C.  for  30  minutes. 

Autolyzed  bacilli  heated  for  30  minutes  at  60°  C.  are  fatal  to  guinea-pigs 
and  mice  in  24-48  hours  when  inoculated  intra-peritoneally  in  doses  of  O'l  c.c. 
The  symptoms  following  the  inoculation  of  toxin  are  very  definite,  the  most 
notable  being  the  glueing  together  of  the  eyelids  and  the  prolonged  (up  to 
9  hours)  narcosis  preceding  death.  Post  mortem,  there  is  hyperaemia  of  the 
lungs,  spleen,  supra-renals,  etc.,  and  frequently  small  necrotic  areas  in  the  liver. 

3.  Agglutination. — The  serum  of  persons  suffering  from  an  infection  with  the 
gaertner  bacillus,  and  the  serum  of  animals  immunized  by  repeated  inoculation 
of  small  sub-lethal  doses  of  the  organism,  have  the  property  of  agglutinating  it. 

In  cases  of  human  infection  the  limits  of  agglutination  are  about  1-250 
to  1—1000.  Human  serums  have  no  action,  or,  at  most,  a  negligible  effect, 
on  the  paratyphoid  B  and  aertrycke  bacilli. 

The  serum  of  immunized  animals  will  agglutinate  the  gaertner  bacillus 
when  diluted  as  much  as  50,000  times,  but  it  has  only  a  very  slight  action 
on  the  paratyphoid  B  and  aertrycke  bacilli :  it  is  therefore  a  simple  matter 
to  distinguish  paratyphoid  B  and  aertrycke  on  the  one  hand  from  gaertner 
on  the  other.  These  facts  may  be  illustrated  by  the  subjoined  table  extracted 
from  Bainbridge  (Jr.  Path,  and  Bact.  xiii.  p.  452). 


AGGLUTINATION  LIMITS  AFTER  INCUBATING  AT  42°  C.  FOR  2  HOURS. 

B.  paratyphoid  B. 

B.  aertrycke. 

B.  gaertner. 

B.  paratyphoid  B,  - 

10,000 

10,000 

<100 

B.  aertrycke,  - 

20,000 

20,000 

<100 

B.  gaertner,    - 

<20 

<20 

50,000 

444  THE  SALMONELLA  GROUP 

SECTION  IV.— ISOLATION  AND   IDENTIFICATION   OF  THE 
BACILLUS. 

The  distribution  of  the  bacillus  in  the  bodies  of  infected  persons  and 
animals  is  the  same  as  in  the  case  of  the  aertrycke  bacillus. 

In  attempting  the  isolation  of  the  gaertner  bacillus  from  material  suspected 
to  contain  it  the  same  methods  will  be  adopted  as  in  the  case  of  other  mem- 
bers of  the  group.  After  preliminary  enrichment  in  dulcite  peptone  water 
the  culture  will  be  plated  on  Conradi-Drigalski's  or  MacConkey's  medium  and  a 
number  of  colourless  colonies  picked  oil  and  tested  as  regards  their  fermenta- 
tion reactions.  If  these  reactions  are  in  agreement  with  those  of  the  gaertner 
bacillus  the  bacilli  must  be  tested  against  a  known  gaertner  serum.  Absorp- 
tion tests  are  unnecessary  in  this  case  because  the  agglutination  reaction  is 
quite  definite. 

4.    P8EUDO-GAERTNER  BACILLI. 

This  term  has  been  adopted  by  Savage  to  describe  certain  organisms  not  uncom- 
monly found  in  food  and  in  the  animal  intestine  and  not  infrequently  present  also  in 
the  human  intestine. 

These  organisms  resemble  the  Salmonella  group  of  bacilli  so  closely  as  to  be, 
culturally  and  biochemically,  almost  indistinguishable  from  them :  they  differ 
from  them  however  in  not  being  agglutinated  by  specific  Salmonella  serums.  It 
follows  from  this  that  pseudo-paratyphoid  B  or  pseudo-aertrycke  bacilli  would  be 
ai>  equally  'correct  designation. 

It  would  seem  probable  that  Savage's  pseudo-gaertner  bacilli  may  be  the  same 
as  the  organisms  referred  to  by  German  observers  as  paratyphoid  C  bacilli. 

There  remain  for  description  three  organisms  discovered  by  various  observers 
in  fatal  diseases  in  mice,  rats  and  parrots  respectively  and  known  as  Bacillus 
typhi  murium.  Danysz's  virus  and  Bacillus  psittacosis.  They  have  now  been 
shown  to  be  identical  either  with  the  aertrycke,  gaertner  or  paratyphoid 
B  bacilli.  The  precise  relationships  of  a  fourth,  Bacillus  icteroides,  have  not 
yet  been  worked  out. 

5.  BACILLUS  TYPHI  MURIUM. 

Lceffler  applied  the  above  description  to  an  organism  which  was  the  infecting 
agent  in  a  fatal  epizootic  among  the  mice  in  his  laboratory. 

The  organism  is  pathogenic  to  mice  (Mus  musculus)  and  field  mice  (Mus  arvicola). 
Loser  investigated  a  similar  epizootic  among  M us  agrarius  :  Merechowsky  and 
Issatchenko  have  also  described  similar  epizootics,  in  one  case  affecting  ground- 
squirrels,  in  the  other  white  rats.  Danysz  recovered  Lceffler's  bacillus  in  an  epizootic 
among  M  us  arvicola. 

Trommsdorf,  whose  observations  have  been  confirmed  by  Mayer  and  Bonhoff  and 
by  Shibayama,  has  shown  that  Loemer's  bacillus  may  infect  man. 

Bonhoff  gave  reasons  for  including  Lceffler's  bacillus  with  the  gaertner  bacillus 
and  the  aertrycke  bacillus  in  the  paratyphoid  group. 

Bainbridge  has  examined  four  strains  of  the  bacillus  typhi  murium  including  one 
from  Lceffler  and  finds  that  the  name  is  applied  to  different  organisms  or  to  impure 
cultures  of  organisms,  thus  :  two  strains  of  the  so-called  bacillus  typhi  murium  were 
cultures  of  the  gaertner  bacillus,  a  third  was  a  mixture  of  the  gaertner  bacillus  and 
the  aertrycke  bacillus  and  a  fourth  a  mixture  of  the  aertrycke  bacillus  and  the 
paratyphoid  B.  bacillus. 

6.    DANYSZ'S  VIRUS. 

From  a  study  of  the  cultural,  agglutination  and  absorption  reactions  of  a  virus 
obtained  from  an  epizootic  among  mice  and  known  as  "  Danysz's  virus,"  Bain- 
bridge  has  shown  that  it  is  a  pure  culture  of  the  bacillus  enteritidis  Gaertner. 


THE  HAT  VIRUSES  445 

Danysz  found  that  the  grey  rat  is  only  moderately  susceptible  to  infection,  and 
that  the  organism  loses  its  virulence  after  a  few  passages  through  rodents  of  this 
species.  He  has  however  succeeded  in  overcoming  the  immunity  in  the  following 
manner  : — the  bacillus  is  first  of  all  grown  in  sealed  ampoules,  then  in  collodion  sacs 
in  the  peritoneal  cavities  of  a  series  of  rats,  then  passed  through  a  mouse  and  finally 
through  a  series  of  rats  (first  young  rats  and  subsequently  older  ones).  Danysz  by 
this  means  obtained  a  strain  which  is  virulent  for  grey  rats  (Mus  decumanus),  black 
rats  and  winter  rats  (Mus  rattus)  and  which  has  been  placed  on  the  market  for  the 
purpose  of  exterminating  rats  by  means  of  the  epizootic  which  it  produces. 

In  view  of  the  researches  of  Bainbridge  which  have  shown  not  only  that  the 
bacillus  typhi  murium  and  Danysz' s  bacillus  are  pathogenic  to  man  but  also  that 
other  so-called  rat  viruses  "  harmless  to  man  and  domestic  animals  "  are  no  other 
than  pure  cultures  or  mixtures  of  the  Salmonella  group,  great  care  is  required  in 
handling  these  viruses.  There  can  be  no  doubt  but  that  human  epidemics  other 
than  those  recorded  by  Shibayama  have  resulted  from  insufficient  care  in  dealing 
with  such  viruses. 

7.    THE  BACILLUS  OF  PSITTACOSIS.1 

Psittacosis  is  an  infectious  disease  of  parrots  and  paroquets  of  which  the  causal 
agent  is  a  bacillus  first  isolated  by  Nocard. 

The  organism  is  a  member  of  the  Salmonella  group  and  is  identical  with  the 
aertrycke  bacillus  (Bainbridge). 

The  bacillus  is  found  in  the  bone  marrow  and  in  the  blood  of  infected  birds  (Nocard). 
Gilbert  and  Fournier  also  isolated  an  organism  similar  to  the  bacillus  of  psittacosis 
from  the  intestines  of  healthy  parrots. 

It  is  said  that  the  disease  may  be  transmitted  to  man  by  contact  with  the  feathers 
of  an  infected  bird  or  with  the  cage  in  which  an  infected  bird  is  confined. 

Parrots  and  paroquets  are  readily  infected  by  experimental  methods..  Sub- 
cutaneous and  intra- muscular  inoculation  often  fail,  but  intra- peritoneal,  intra- 
venous and  intra-tracheal  inoculation,  and  ingestion,  set  up  an  infection  which 
proves  fatal  in  a  few  days.  Infected  birds  sit  huddled  up  and  motionless  on  their 
perches  with  their  feathers  ruffled  and  wings  drooping.  They  suffer  from  diarrhoea, 
refuse  their  food  and  are  in  a  constant  state  of  drowsiness. 

Mice,  fowls  and  pigeons  can  also  be  infected.  Rabbits  and  especially  guinea-pigs 
are  more  immune. 

The  bacillus  of  psittacosis  being  identical  with  the  aertrycke  bacillus  it  follows 
that  the  same  methods  of  isolation  and  identification  are  applicable  to  the  former  as 
to  the  latter. 

8.    BACILLUS  ICTEEOIDES. 

This  bacillus  was  isolated  from  cases  of  yellow  fever  and  for  a  time  was  considered 
to  be  the  cause  of  the  disease,  which  is  however  now  known  to  be  due  to  a  filter- 
passing  organism.  The  bacillus  icteroides  is  a  member  of  the  Salmonella  group,  and 
has  been  proved  to  be  identical  culturally  with  Salmon  and  Smith's  hog  cholera  bacillus 
(bacillus  aertrycke).  No  absorption  tests  however  appear  to  have  been  carried  out 
so  that  it  is  uncertain  whether  it  is  a  paratyphoid  B,  an  aertrycke  or  a  gaertner 
bacillus. 

1  L.  Psittacus  and  Gr.  ^trra^os,  parrot. 


CHAPTER   XXVIII. 
THE  PASTEURELLA  GROUP  OF  BACILLI. 

Introduction. 

1.  Pasteurella  gallinse,  p.  447. 
Section  I. — Experimental  inoculation,  p.  448. 
Section  II. — Morphology,  p.  449. 

Section  III. — Biological  properties,  p.  451. 

Section  IV. — The  isolation  and  identification  of  the  bacillus,  p.  452. 
Similar  organisms  in  epizootics  among  other  birds,  p.  452. 

2.  Pasteurella  cuniculi,  p.  453. 

3.  Pasteurella  suis,  p.  454. 

4.  Pasteurella  bovis,  p.  455. 

5.  Pasteurella  ovis,  p.  456. 

6.  Pasteurella  capree,  p.  456. 

7.  Pasteurella  equi,  p  456. 

8.  Pasteurella  canis,  p.  457. 

M'Gowan's  bacillus  of  distemper,  p.  459. 

9.  Immunization  with  polyvalent  vaccines,  p.  459. 

SINCE  the  discovery  by  Pasteur  of  the  organism  which  is  the  cause  of  the 
disease  known  as  fowl  cholera,  a  similar  organism  has  been  isolated  by 
many  observers  from  epizootics  affecting  widely  different  species  of  animals. 
The  similarity  between  the  organisms  obtained  from  different  animal 
species  soon  attracted  attention.  Hueppe  classified  in  one  group  the 
epizootic  diseases  affecting  fowls,  pigs,  rabbits,  ferrets,  cattle  and  wild 
animals,  and  described  them  as  the  "  hcemorrhagic  septiccemias."  Nocard 
and  Leclainche  proceeding  further  in  the  generalization,  affirmed  that  the 
causal  organisms  of  all  these  diseases  were  merely  varieties  of  one  and  the 
same  organism  to  which  they  applied  the  term  the  "  ovoid  bacterium  "  (la 
bacteridie  ovoide)  :  but  to  perpetuate  the  discovery  of  the  organism  of  fowl 
cholera  by  Pasteur,  Toni  and  Trevisan  appropriately  suggested  the  use  of 
the  term  Pasteurella  in  place  of  bacteridie  ovoide.  Lignieres  from  a  study  of 
the  organisms  isolated  from  birds,  pigs,  cattle,  etc.,  came  to  the  conclusion 
that  varieties  of  the  Pasteurella  could  be  distinguished  by  means  of  their 
pathogenic  properties,  and  recent  researches — especially  those  of  Chamber- 
land  and  Jouan — have  justified  this  view.  Consequently,  as  Nocard  held, 
the  organisms  isolated  from  different  animal  species  must  be  regarded  as 


THE  AVIAN  PASTEURELLA  447 

varieties  of  the  same  bacillus,  and  the  conclusion  arrived  at  is  in  short  this  : 
that  there  is  one  Pasteurella,  which  can  pass  from  one  animal  species  to 
another,  and  which  by  adaptation  in  one  species  can  produce  a  disease  peculiar 
to  that  species. 

Chamberland  and  Jouan  showed  that  the  avian  pasteurella  could  naturally  infect 
pigs  and  the  swine  pasteurella  fowls. 

By  passage  through  rabbits  in  the  laboratory  the  ovine  pasteurella  acquires  the 
properties  of  the  avian,  and  similarly,  by  passage  through  guinea-pigs,  young 
chickens  and  fowls  the  swine  pasteurella  acquires  the  characteristics  of  the  avian 
pasteurella. 

Immunization  with  one  variety  establishes  immunity  against  that  as  well  as 
against  the  other  varieties  :  thus,  rabbits  vaccinated  with  the  swine  pasteurella  are 
at  the  same  time  immunized  against  the  fowl  pasteurella,  and  so  on. 

A  description  of  the  disease  (Pasteur ellosis]  as  it  occurs  in  different  animals 
will  now  be  given,  but  it  must  be  distinctly  understood  that  this  plan  of 
dealing  with  the  subject  is  adopted  merely  for  convenience,  and  does  not 
imply  any  doubt  as  to  the  truth  of  Nocard's  view  of  the  specific  identity  of 
the  organisms  found  in  the  different  species  of  animals. 

Characteristics  common  to  the  pasteurella  group. — Organisms  of  the  pas- 
teurella group  are  non-motile  cocco-bacilli,  gram-negative,  very  pleomorphic 
and  staining  more  deeply  at  the  ends  than  in  the  centre  [cf.  B.  pestis].  They 
do  not  form  spores,  do  not  liquefy  gelatin  nor  coagulate  milk,  they  give  no 
visible  growth  on  potato,  are  primarily  aerobic  but  can  be  grown  anaerobically. 
They  give  rise  in  culture  to  a  peculiar  and  characteristic  odour. 

According  to  Lignieres  these  organisms  do  not  produce  indol  in  culture,  but  old 
cultures  of  the  bacillus  of  fowl  cholera  (the  avian  pasteurella)  certainly  contain 
indol. 

[The  pasteurelloses  and  plague  in  animals. — Attention  must  be  drawn  to 
the  close  resemblance  which  exists  between  the  bacilli  of  the  pasteurella  group 
on  the  one  hand  and  the  plague  bacillus  on  the  other,  and  also  to  the  similarity 
of  the  lesions  naturally  produced  by  these  organisms.  Not  only  are  these 
bacilli  very  much  alike,  indeed  almost  identical,  in  their  morphological 
appearances  and  cultural  characteristics,  but  the  naked  eye  lesions  produced 
in  naturally  infected  animals  are  also  very  similar.  Mistakes  are  therefore 
certain  to  be  made  unless  in  every  case  the  causal  organism  is  isolated  and 
differential  tests  applied. 

[Culturally  the  plague  bacillus  is  most  readily  differentiated  from  bacilli 
of  the  pasteurella  group  by  an  observation  of  the  growth  in  MacConkey's 
sodium  taurocholate  medium  (p.  412)  containing  the  following  carbohydrates — 
glucose,  Isevulose,  mannite  and  galactose.  The  plague  bacillus  grows  in  all 
these  media,  producing  acid  but  no  gas.  The  bacilli  of  the  pasteurella  group 
do  not  grow  in  MacConkey's  medium  (Indian  Plague  Commission). 

[The  formation  of  stalactites  in  broth  culture  is  not  a  feature  peculiar  to 
the  plague  bacillus  but  is  possessed  also  by,  at  any  rate,  many  of  the 
pasteurella  bacilli. 

[Animal  inoculation  will  further  assist  the  differential  diagnosis  (cf.  Plague).  ] 


1.    PASTEURELLA  GALLING. 

(The  bacillus  of  fowl  cholera.) 

Pasteur  was  the  first  to  describe  the  avian  pasteurella,  though  it  had 
previously  been  seen  by  Moritz,  Perron9ito,  and  by  Toussaint. 

Fowl  cholera  (fowl  plague,  fowl  septicaemia,  fowl  typhoid)  is  an  epizootic 
disease  of  the  Gallinaceae  (fowls,  pheasants,  guinea-fowl,  turkeys  and  pigeons) 


448  THE  PASTEURELLA  GROUP 

and  Palmipedse  (ducks  and  geese).  Rabbits  though  very  susceptible  to 
experimental  infection  are  rarely  attacked  by  the  epizootic  disease. 

The  course  of  the  disease  may  be  rapid  and  its  onset  sudden.  Usually  however 
it  is  not  so  acute,  and  the  animals,  after  being  miserable  and  drowsy  and  suffering 
from  an  attack  of  diarrhoea,  often  hsemorrhagic  in  character,  die  in  5-7  days.  The 
sick  birds  do  not  feed,  they  droop  their  wings,  their  feathers  are  dull  and  bristling 
and  their  combs  black.  Towards  the  end  of  the  outbreak  the  cases  become  less 
severe  and  some  of  the  birds  recover. 

Infection  takes  place  via  the  alimentary  canal  by  means  of  food  contaminated 
by  the  dejecta  of  birds  suffering  from  the  disease.  The  organism  is  found  in  the 
blood,  internal  organs  and  intestines  in  acute  cases,  but  it  is  impossible  to  find  it  in 
chronic  cases. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

Animals  may  be  infected  by  inoculating  them  with  a  few  drops  of  a 
24-hour-old  culture,  or  with  the  blood  of  an  animal  which  has  just  died  of 
fowl  cholera,  either  sub-cutaneously  or  intra-peritoneally  or — in  the  case  of 
birds — in  the  pectoral  muscle. 

To  produce  infection  in  animals  by  feeding  the  food  may  be  watered  with 
a  virulent  culture  or  they  may  be  fed  upon  the  tissues  of  an  animal  which  has 
died  of  the  disease. 

1.  Susceptible  animals. — Birds  generally  and  especially  small  birds  (sparrows, 
etc.)  are  very  susceptible  ;  they  may  be  infected  in  various  ways,  though 
feeding  often  fails  to  produce  the  disease. 

Rabbits  are  particularly  susceptible  to  the  fowl  pasteurella,  and  may  be 
easily  infected  by  feeding  or  by  sub-cutaneous  inoculation.  They  die  in  the 
very  early  stages  of  the  disease,  before  diarrhoea  has  had  time  to  manifest 
itself,  though  on  post  mortem  examination  the  intestine  is  found  to  be  full 
of  liquid  matter.  This  fact  affords  an  explanation  of  the  rarity  of  the  epizootic 
disease  among  rabbits,  the  excreta  being  the  ordinary  vehicle  of  infection. 

Post  mortem  the  stomach  is  distended,  the  blood  black  and  hsemolyzed  ; 
there  are  numerous  effusions  of  blood  and  the  pleurae  on  both  sides  are 
affected. 

Mice  and  rats  are  very  susceptible,  and  the  ground  squirrel  should  be 
mentioned  among  rodents  susceptible  to  the  disease.  It  was  at  Metchni- 
koff's  suggestion  that  the  bacillus  of  fowl  cholera  was  used  to  start  true 
epizootics  among  ground  squirrels  in  order  to  diminish  their  numbers  in 
parts  of  Southern  Russia  which  were  infested  with  them. 

Guinea-pigs  are  fairly  immune.  The  inoculation  of  a  moderate  dose  of  a 
virulent  culture  into  the  sub-cutaneous  tissues  does  not  kill  the  animal,  but 
merely  produces  an  abscess ;  the  pus  from  the  abscess  contains  but  few  micro- 
organisms but  is  nevertheless  virulent  for  fowls.  Guinea-pigs  however 
generally  succumb  as  a  result  of  the  injection  of  a  virulent  culture  into  the 
peritoneal  cavity  ;  and  if  a  given  culture  be  passed  from  animal  to  animal 
in  this  way  its  virulence  can  be  increased  sufficiently  to  kill  a  guinea-pig  on 
sub-cutaneous  inoculation. 

Dogs  and  cats  are  only  slightly  susceptible  ;  sub-cutaneous  inoculation 
produces  a  swelling  which  quickly  subsides  and  the  animal  recovers  ;  intra- 
venous inoculation  is  followed  by  more  severe  results  and  may  prove  fatal. 
By  passage  through  a  series  of  dogs — or  cats — the  virulence  of  the  organism 
for  the  species  is  considerably  increased,  and  sub-cutaneous  inoculation  will 
then  prove  fatal. 

Pigs  are  only  slightly  susceptible  to  sub-cutaneous  inoculation  but  often 
die  after  intra- venous  inoculation. 


THE  FOWL  PASTEURELLA  449 

Sheep  are  susceptible,  and  succumb  rapidly  to  the  intra- venous  inoculation 
of  a  highly  virulent  culture  ;  with  a  less  virulent  virus  the  animal  dies  only 
after  an  interval  of  some  days,  having  in  the  meantime  suffered  from  a 
multiple  purulent  arthritis. 

2.  Symptoms  and  lesions. — The  symptoms  in  the  fowl  will  be  described,  as 
this  is  the  animal  most  frequently  used  for  experimental  purposes.  The 
disease  in  the  rabbit  has  already  been  described. 

A  few  drops  of  a  virulent  culture  will  kill  fowls  in  12-30  hours.  In  fulminat- 
ing cases  there  are  practically  no  symptoms  ;  if  however  the  animals  live  long 
enough  the  symptoms  are  the  same  as  in  the  spontaneous  disease  : — at 
first  the  animal  is  unsteady  and  swells  itself  out,  its  feathers  are  ruffled  and 
its  comb  black,  then  purging  begins  and  the  dejecta  are  mucous  or  blood- 
stained :  finally  the  fowl  becomes  drowsy  and  comatose  and  death  follows 
a  series  of  convulsions.  When  inoculated  with  a  less  virulent  virus  the 
animal  may  recover  from  the  disease  or  die  after  the  lapse  of  several  days 
with  cachexia  and  arthritis  of  its  hind  limbs. 

Post  mortem,  at  the  site  of  inoculation,  there  is  a  very  small  quantity 
of  blood-stained  oedema  rich  in  bacilli.  If  the  inoculated  virus  were  only 
slightly  virulent  the  oedema  is  more  marked,  and  when  the  disease  lasts 
for  several  days  the  infiltration  is  gelatinous,  the  pectoral  muscle  around 
the  site  of  inoculation  is  swollen  and  yellowish  in  colour  and  may  even  have 
a  lardaceous  appearance  (necrosis).  The  blood  is  black,  coagulates  feebly 
and  has  the  appearance  of  being  hsemolyzed,  and  contains  the  pasteurella 
in  enormous  numbers.  The  sub-cutaneous  cellular  tissues,  the  serous  mem- 
branes and  internal  organs  all  show  haemorrhages  :  the  lungs  contain  foci  of 
infiltration,  the  liver  is  large,  yellowish  and  friable,  and  the  spleen  swollen 
and  softened.  These  last-named  lesions  are  not  however  constant.  The 
pericardium  contains  a  clear  serous  fluid  sometimes  blood-stained  or  gela- 
tinous ;  and  a  similar  effusion  is  found  in  the  pleural  cavities  in  rabbits  (birds 
do  not  possess  a  pleural  cavity).  The  muscles  and  the  heart  are  only  affected 
if  the  disease  has  lasted  several  days  ;  in  that  case  they  are  soft  and  yellowish 
like  a  dead  leaf.  The  intestine  presents  the  lesions  of  enteritis,  the  mucous 
membrane  being  injected  and  ulcerated  in  patches.  The  intestinal  contents 
are  fluid  and  sometimes  blood-stained.  When  the  virus  inoculated  is  only 
slightly  virulent  and  death  is  delayed,  arthritis 
of  the  joints  of  the  posterior  limbs  can  often  be 
demonstrated  on  post  mortem  examination  (Lig- 
nieres). 

As   usual,   the   lesions   are  more  marked  the 
longer  the  animal  lives. 


SECTION  II.-  MORPHOLOGY. 
1.  Microscopical  appearance. 

The  fowl  pasteurella  is  a  small  bacillus  or  cocco- 
bacillus.     The  length  does  not  exceed  0'5-1'25/A 
and  the  breadth  0'25-0'40/a.     In  unstained  pre- 
parations the  organism   has  the  appearance  of    ^th,  Reich.) 
more  or  less  elongated  points  refractile  in  the 

centre  and  often  arranged  in  pairs  :  it  exhibits  active  Brownian  movement 
but  no  movement  of  translation.  In  stained  preparations,  which  are  the 
best  for  a  study  of  the  morphology  of  the  bacillus,  it  is  seen  to  be  oval 
in  shape  with  rounded  ends,  and  when  lightly-stained  preparations  (thionin) 


450  THE   PASTEURELLA   GROUP 

are  examined  a  small  unstained  refractile  area  is  seen  in  the  centre.  In  rapidly 
growing — young — cultures  of  the  highly  virulent  strains,  the  rounded  forms 
predominate  and  the  appearance  is  suggestive  of  diplococci ;  in  older  or  in 
less  virulent  cultures  the  elongated,  bacillary  forms  are  chiefly  found. 

The  fowl  pasteurella  does  not  form  spores. 

Staining  methods. — The  bacillus  is  easily  stained  with  the  ordinary  dyes, 
and  is  gram-negative. 

(a)  Cultures. — Stain   with   carbol-thionin,    Kiihne's   carbol-blue   or   dilute 
carbol-fuchsin. 

(b)  Scrapings  of  organs  and  blood  films.     1 .  Simple  staining.— Stain  for  a  few 
minutes  with  carbol-blue  or  carbol-thionin,  wash,  dry  and  mount.     The  leuco- 
cytes, the  nuclei  of  the  red  cells  and  the  bacilli  are  stained  deep  blue  or  violet. 

2.  Double  staining. — Drop  a  little  1  per  cent,  aqueous  solution  of  eosin  on 
a  blood  film,  leave  for  2  or  3  minutes,  wash,  then  pass  through  carbol-blue, 
wash  again,  dry  and  mount.  This  method  gives  exceedingly  pretty  prepara- 
tions ;  the  cytoplasm  of  the  red  cells  is  stained  red  by  the  eosin  while  their 
nuclei  and  the  micro-organisms  are  blue  (fig.  225).  Unfortunately  the 
technique  is  a  little  difficult :  the  action  of  the  blue  must  be  carefully 
watched  under  the  microscope  and  the  dye  washed  off  as  soon  as  the 
differentiation  is  complete. 

(c)  Sections. — Nicolle's  tannin  method  should  be  used. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  fowl  pasteurella  is  primarily  an  aerobic  organism. 
It  will  also  grow  in  media  deprived  of  oxygen,  but  anaerobic  cultures  are 
always  very  poor  and  can  only  be  obtained  under  certain 
conditions,  e.g.  by  sowing  freely  in  serum  broth.  In  aerobic 
cultures  the  organism  grows  feebly  between  20°  and  25°  C. ; 
the  optimum  temperature  is  from  35°-39°  C. 

The  most  useful  media  are  chicken-  or  veal-broth  made 
neutral  or  slightly  alkaline,  and  especially  broths  containing 
serum. 

Cultures  in  peptone  broth  produce  indol  but  only  after 
incubating  for  about  a  fortnight  (Porcher  and  Panisset). 

Characters  of  growth.  Broth.— At  37°  C.  growth  takes 
place  rapidly  in  chicken-broth.  A  slight  cloudiness  is 
visible  in  about  10  hours  which  increases  for  the  next 
10-12  hours,  then  the  growth  deposits  in  the  form  of  a 
scanty  precipitate  leaving  the  medium  clear.  Growth 
ceases  in  about  a  week. 

The  virulence  of  the  culture  reaches  its  maximum  in  about  24 
hours ;  at  this  stage  the  rounded  forms  predominate,  while  in 
older  cultures  the  long  forms  are  the  more  numerous.  When 
growth  has  ceased,  the  precipitate  consists  of  granular  debris 
in  which  no  definite  structure  can  be  made  out ;  if  sown  on  a  fresh 
medium,  however,  it  will  for  some  time  give  rise  to  virulent 
cultures. 

FIG.  226.— Pasteur-       Agar. — Growth  is  rapid  at  37°  C. ;  a  thin  white  glistening 

eiia    gaiiincK.     stab   streak  is  formed,  thicker  in  the  centre  than  at  the  edges. 

^e  j  >   If  a  drop  of  blood  be  smeared  on  the  surface  of  the  agar, 

single  colonies  will  usually  be  obtained  which  are  at  first 

transparent  and  bluish  and  later  semi-opaque. 

Coagulated  serum. — The  growth  on  this  medium  has  the  same  appearance 
as  the  growth  on  agar. 


THE  FOWL  PASTEURELLA  451 

Gelatin. — At  22° -23°  C.  growth  is  both  slow  and  minimal  in  amount. 
Stab  culture  gives  a  thin  white  line  spreading  out  a  little  on  the  surface  like 
a  nail-head,  the  growth  is  very  poor  in  the  depth.     Stroke  culture  produces  a 
very  fine  whitish  line  which  appears  blue  by  transmitted  light.     The  gelatin  is 
not  liquefied. 

Potato  :   Yeast  extract. — No  visible  growth  occurs  on  these  media- 
Milk. — Growth  takes  place  without  coagulation  of  the  medium. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Vitality  and  virulence. 

The  fowl  pasteurella  is  a  very  delicate  organism  and  rapidly  dies  out  in 
culture.  It  is  easily  killed  by  drying,  or  by  heating  to  55°  C.,  as  well  as  by 
antiseptics  or  very  dilute  acids. 

Cultures  in  liquid  media  are  more  resistant  than  those  on  solid  media,  but 
a  broth  culture  dies  out  in  about  6  weeks  or  2  months,  and  while  alive  rapidly 
loses  its  virulence.  The  attenuation  is  due  to  the  action  of  the  oxygen  of 
the  air  ;  infected  blood  stored  in  sealed  capsules  keeps  its  virulence  for  a 
long  time,  and  a  virulent  culture  may  be  similarly  preserved  by  incubating 
it  in  broth  for  18  hours  and  then  sealing  it  up. 

2.  Attenuation— Vaccination. 

The  virulence  of  broth  cultures  kept  at  37°  C.  is  very  materially  reduced 
at  the  end  of  a  fortnight ;  such  a  culture  inoculated  into  a  series  of  animals 
does  not  kill  more  than  two  or  three  out  of  every  ten  inoculated.  A  few 
days  later  still  the  culture  has  almost  entirely  lost  its  virulence  :  when  inocu- 
lated into  fowls  it  does  not  set  up  a  fatal  disease  but  merely  a  temporary 
indisposition,  and  if  the  pectoral  muscle  be  the  site  of  inoculation  only  a 
local  lesion  (gelatinous  oedema,  muscular  sequestrum,  or  necrosis)  results. 
The  virulence  of  the  attenuated  virus  though  slight  is  however  fixed,  and  by 
storing  it  in  sealed  capsules  a  culture  with  this  character  can  be  preserved  : 
cultures  of  different  degrees  of  virulence  may  also  be  kept  under  similar 
conditions. 

It  is  always  possible  to  make  these  cultures  fully  virulent  by  passage  through 
sparrows  ;  a  virus  which  will  not  kill  fowls  is  still  fatal  to  sparrows  and  after  a  few 
passages  will  have  fully  recovered  its  virulence,  so  that  on  inoculation  into  fowls 
death  results  in  a  few  hours. 

It  was  when  working  with  the  bacillus  of  fowl  cholera  in  1878  that  Pasteur 
discovered  that  organisms  might  lose  their  virulence,  which  observation  was 
the  basis  of  the  discovery  of  vaccination  with  micro-organisms. 

A  fowl  which  has  suffered  from  a  mild  attack  of  the  disease  following  the 
inoculation  of  an  attenuated  virus  is  after  recovery  no  longer  susceptible  to 
the  disease  ;  it  can  be  inoculated  with  the  most  virulent  strains  without 
suffering  any  harm.  Prophylactic  vaccination  is  effected  in  practice  by 
inoculating  into  the  tip  of  the  wing  first  a  very  attenuated  virus  and  a  few 
days  later  a  second  stronger  vaccine.  The  animal  is  then  immune  for  about 
a  year. 

3.  Toxin. 

Filtered  broth  cultures  when  inoculated  into  fowls  produce  a  disease 
characterized  by  weakness  and  drowsiness  from  which  the  animals  always 
recover  and  which  confers  on  them  a  certain  degree  of  immunity  (Pasteur). 
Fowls  which  have  been  vaccinated  with  the  micro-organism  are  still 
susceptible  to  the  toxin. 


452  THE   PASTEURELLA  GROUP 

Bisanti  succeeded  in  immunizing  rabbits  by  inserting  a  culture  of  the 
bacillus  in  a  collodion  sac  beneath  the  skin  or  in  the  peritoneal  cavity. 
Rabbits  treated  in  this  way  were  at  the  end  of  20  days  unaffected  when 
fed  on  virulent  cultures. 


SECTION  IV.— THE   ISOLATION  AND   IDENTIFICATION   OF   THE 

ORGANISM. 

The  blood,  the  internal  organs  and  the  intestinal  contents  should  provide 
the  material  for  bacteriological  examination. 

1.  Microscopical  examination. — Films  prepared  with  blood,  scrapings  of  the 
internal  organs  or  contents  of  the  intestinal  canal  should  be  stained  with 
carbol-thionin  or  carbol-blue  and  by  Gram's  method.     Sections  of  the  internal 
organs  are  particularly  interesting  and  show  the  capillaries  engorged  with 
bacilli. 

2.  Cultures. — Tubes  of  ordinary  broth  or,  better,  veal  broth,  should  be  sown 
with  blood  from  the  heart  or  with  a  loopful  of  pulp  from  the  spleen  or  liver. 

At  the  post  mortem  a  small  stock  of  blood  can  be  collected  and  stored  for  sowing 
cultures  later.  For  this  purpose  aspirate  the  blood  into  a  pipette  (p.  75)  and  then 
seal,  first  the  fine  end  then  the  constricted  portion.  Culture  media  sown  with 
blood  stored  in  this  way  will  even  many  months  later  give  growths  of  virulent 
organisms. 

3.  Inoculations. — Inoculation  should  be  made  for  preference  into  a  fowl 
or  a  rabbit.     Blood  from  the  heart  or  spleen  pulp  or  better  still  a  few  drops 
of  a  recent  culture  in  fowl  broth  or  serum  broth  may  be  used. 

Epizootics  similar  to  fowl  cholera  in  other  birds. 

The  bacillus  of  duck  cholera. — Cornil  and  Toupet  described  an  epizootic  among 
ducks  which  was  characterized  by  diarrhoea,  often  blood-stained,  and  by  feebleness 
and  drowsiness.  The  disease  is  due  to  a  small  bacillus  very  similar  to  that  of  fowl 
cholera,  from  which  it  is  only  differentiated  by  the  following  characteristics. 

1.  It  grows  on  potato  giving  rise  to  a  scanty  yellowish  growth. 

2.  Cultures  of  the  organism  which  are  virulent  for  ducks  are  almost  harmless 
to  fowls  and  pigeons  and  are  only  fatal  to  rabbits  when  inoculated  in  large  doses. 

Lignieres  does  not  include  this  organism  in  the  pasteurella  group. 

Klein's  [so-called]  bacillus  of  grouse  disease. — A  small,  motile  bacillus  measuring 
0*6— 1'5/x  long,  gram-negative,  giving  copious  growths  aerobically  on  broth,  agar, 
gelatin  (without  liquefaction)  and  potato,  coagulating  milk  and  producing  indol. 
[This  organism  appears  to  be  the  colon  bacillus  which  invades  the  tissues  of  the 
birds  after  death.] 

This  bacillus  should  not  therefore  be  included  in  the  pasteurella  group  as  defined 
by  Lignieres. 

'The  bacillus  of  wood-pigeon  disease  (Leclainche). — A  motile  bacillus  morpho- 
logically identical  with  Klein's  grouse  disease  bacillus.  It  grows  on  potato  and  is 
virulent  for  pigeons,  rabbits  and  guinea-pigs. 

The  bacillus  of  infectious  enteritis  of  fowls  (Klein). — The  symptoms  of  the  disease 
are  very  similar  to  those  of  fowl  cholera.  The  bacillus  presents  the  typical  charac- 
teristics of  the  pasteurella  group,  is  non-motile,  gram-negative,  does  not  liquefy 
gelatin  and  does  not  grow  on  potato. 

The  bacillus  of  epizootic  dysentery  in  fowls  and  turkeys  (Lucet). — This  organism 
should  be  distinguished  from  the  preceding  (Lignieres).  It  grows  on  potato. 

The  bacillus  of  coscoroba  swan  disease. — Tretrop  described  a  disease  which  broke 
out  among  the  Coscoroba  swans  in  the  Zoological  Gardens  at  Antwerp. 

The  disease  has  the  symptoms  of  duck-cholera,  intestinal  disturbances  being  the 
more  prominent  feature  ;  it  does  not  attack  other  species  of  swans,  teal,  ducks,  nor 
geese  which  have  been  in  contact  with  the  sick  birds.  The  cause  of  the  disease  is 
a  small  cocco-bacillus,  indifferently  aerobic,  similar  to  the  fowl  pasteurella  in  appear- 


THE  RABBIT  PASTEURELLA 


453 


ance  and  gram-negative.  It  is  pathogenic  for  mice  and  swallows,  guinea-pigs  are 
only  slightly  susceptible,  fowls  and  ducks  are  immune. 

Tretrop  was  able  with  difficulty  to  vaccinate  mice  against  the  disease  by  treating 
them  with  cultures  attenuated  by  heat  (10  minutes  at  58°  C.). 

The  bacillus  of  hsemorrhagic  septicaemia  of  ducks  and  fowls  (Rabieaux). — This 
bacillus  has  all  the  characteristics  of  the  fowl  Pasteurella.  It  is  an  oval  bacterium, 
non-motile,  pleomorphic,  gram-negative  and  does  not  grow  on  potato.  It  is  patho- 
genic for  ducks,  fowls,  pigeons,  rabbits,  guinea-pigs  and  white  rats. 

The  repeated  inoculation  of  filtered  cultures  of  this  bacillus  or  of  cultures  heated 
to  60°  C.  renders  rabbits  and  guinea-pigs  immune  to  the  sub-cutaneous  inoculation 
of  living  and  virulent  cultures,  infected  blood  and  other  material. 


2.   PASTEURELLA  CUNICULI. 
(The  bacillus  of  rabbit  septicaemia). 

A  large  number  of  dissimilar  affections  due  to  widely  different  organisms 
have  been  classed  together  under  the  head  of  "rabbit  septicaemia,"  but  many 
of  these  septicaemias  are  undoubtedly  caused  by  an  organism  having  all  the 
characteristics  of  the  pasteurella  (P.  cuniculi)  first  described  by  Th.  Smith 
and  more  fully  later  by  Thoinot  and  Masselin. 

[C.  J.  Martin  and  Rowland  have  recently  found  that  both  plague  (B. 
pestis)  and  rabbit  septicaemia  (P.  cuniculi)  may  be  encountered  among  rabbits 
in  the  same  neighbourhood.  "  The  co-existence  of  this  latter  disease  [rabbit 
septicaemia]  indicates  the  need  for  care  in  the  diagnosis  of  plague  among 
these  rodents,  as  in  the  organs  the  cocco-bacilli  of  rabbit  septicaemia  may  be 
microscopically  indistinguishable  from  Bacillus  pestis."  x] 

The  bacillus  described  by  Eberth  and  Mandry  and  isolated  by  them  during  an 
epizootic  among  rabbits  is  an  oval-shaped  motile  organism,  growing  on  potato, 
coagulating  milk  and  producing  indol.  These  characteristics  should  exclude  it 
from  the  Pasteurella  group  which  in  all  other  respects  it  resembles.  The  bacillus  of 
ferret  septicaemia  of  Eberth  and  Schimmelsbuch  is  also  wanting  in  most  of  the 
characteristics  of  a  typical  pasteurella. 

The  bacillus  described  by  Lucet  in  a  "  new  septicaemia  of  rabbits  "  in  1889  should 
apparently  be  included  with  the  pasteurella,  and  also  a  micro-organism  described 
by  Lefebre  and  Gautier  in  a  septicaemia  similar  to  that  of  Eberth. 

1.  Experimental  inoculation.  —  Rabbits  and  guinea-pigs  are  equally 
susceptible  to  inoculation  with  the  rabbit  pasteurella  ;  all  birds  are  also 
susceptible.  A  young  broth  culture  or  material 
(blood,  spleen  or  liver  tissue)  from  an  animal 
recently  dead  of  the  disease  may  be  used  ;  intra- 
peritoneal  inoculation  is  more  rapidly  fatal  than 
sub-cutaneous  inoculation. 

[Martin  and  Rowland  found  rats  and  guinea- 
igs  to  be  unaffected  by  a  dose  of  culture  which 
illed  large  rabbits  in  less  than  18  hours.  ] 

Symptoms  appear  on  an  average  about  20 
hours  after  inoculation.  The  animal  is  weak 
and  refuses  its  food,  respiration  is  quickened, 
locally  there  is  a  swelling  at  the  site  of  inocula- 

•V        ,  -IT,  -i        FIG.    227.  —  Pasteurella  cumcuh. 

tion,  diarrhoea  supervenes  and  later  the  animal   on  the  left  a  film  from  sparrow's 


«.*£*?*  a  fllm  from  the 


becomes  comatose  and  dies. 

Post  mortem,  the  venous  system  is  engorged 
with  thick,  dark-coloured  blood,  the  trunk  muscles  are  of  a  reddish  purple 
colour,  the  abdominal  cavity  contains  a  large  quantity  of  thick,  blood-stained 
1  Report  Medical  Officer,  Local  Government  Board,  1910-11,  Cd.  5939. 


454  THE  PASTEURELLA  GROUP 

fluid,  the  lungs  are  congested  and  seem  to  float  in  a  blood-stained  effusion, 
and  the  pericardium  is  full  of  fluid  which  is  also  occasionally  blood-stained. 

2.  [Post-mortem  appearances  in  naturally  infected  rabbits  and  in  spontaneous 
plague  in  rabbits,     (a)  Rabbit  septicaemia. — In  a  case  observed  by  C.  J.  Martin  and 
Rowland  the  right  superficial  inguinal  gland  was  red  and  swollen,  and  the  vessels 
in  the  neighbourhood  congested.     The  skin  generally  was  injected,  and  both  skin 
and  peritoneal  lining  had  a  pink  flush.     The  spleen  was  enlarged  and  tense,  and  the 
liver  mottled.     The  pleurae  and  pericardium  were  full  of  fibrinous  exudation,  the 
heart  being  adherent  to  the  parietal  pericardium  and  the  lungs  to  the  pleurae.     There 
was  double  pneumonia.     The  organism  was  found  in  the  pleural  exudate,  lungs, 
spleen  and  enlarged  glands. 

[(&)  Plague. — In  another  rabbit  from  the  same  neighbourhood  these  observers 
found  a  plague-infected  rabbit  which  showed  the  following  lesions :  a  typical  sub- 
maxillary  bubo,  and  marked  injection  of  the  vessels  in  the  skin.  The  spleen  was 
much  enlarged  tense  and  of  a  purplish  colour.  The  peritoneum  and  pleurae  con- 
tained blood-stained  fluid.  The  left  lung  was  congested  but  not  consolidated. 
The  intestines  were  matted  together  by  recent  lymph. 

[In  a  second  rabbit  the  only  lesion  was  a  greatly  enlarged  spleen  full  of  nodules.] 

3.  Morphology. — Microscopically  the  organism  is  similar  to  that  of  fowl 
cholera   (q.v.)    [and    may   be   indistinguishable   from    Bacillus  pestis].     Its 
growth  on  gelatin  is  white,  slightly  viscous  and  rather  more  abundant  than 
that  of  the  fowl  cholera  bacillus. 


3.    PASTEURELLA  8UI8. 

(The  bacillus  of  contagious  pneumonia  of  pigs  or  swine  plague). 
Ger.  Schweineseuche.     Fr.  Pasteurellose  du  pore. 

The  contagious  pneumonia  of  pigs,  known  in  America  as  "  Swine  plague  " 
and  in  Germany  as  "  Schweineseuche,"  and  caused  by  an  organism  of  the 
pasteurella  group,  must  be  carefully  distinguished  from  Hog  Cholera  1  (Chap. 
LXIV.)  with  which  it  was  for  a  long  time  confused. 

Clinically  the  differential  diagnosis  is  very  difficult  and  sometimes  impossible, 
and  further,  as  Karlinski  has  shown,  an  animal  may  suffer  from  the  two  diseases 
at  the  same  time.  Bacteriologically,  the  micro-organisms  are  very  different ;  the 
organism  of  swine  plague  belongs  to  the  pasteurella  group  and  is  closely  allied  to 
the  bacillus  of  fowl  cholera,  while  the  Hog  Cholera  bacillus  or  Bacillus  suipestifer 
belongs  to  the  Salmonella  group  (p.  431).  It  should  be  stated  however  that  Hutyra, 
as  a  result  of  his  investigations,  inclines  to  the  belief  that  hog-cholera  and  swine 
plague  (Schweineseuche)  are  one  and  the  same  disease  and  due  to  an  invisible 
micro-organism. 

[It  would  seem  also  that  the  pasteurella  infection  of  pigs  may  be  mistaken 
for  an  infection  with  Bacillus  pestis  (cf.  p.  461).] 

Experimental  inoculation. — The  virulence  of  the  swine  pasteurella  is  ex- 
ceedingly variable  but  if  low  is  easily  increased  by  passage.  Mice  and  rabbits 
soon  succumb  after  being  inoculated  sub-cutaneously  with  a  virulent  strain ; 
guinea-pigs  are  less  susceptible  ;  in  pigeons  the  inoculation  of  O5  c.c.  of  a 
virulent  culture  into  the  muscles  proves  fatal.  Fowls  are  more  resistant, 
but  the  inoculation  of  a  virus  which  has  been  increased  in  virulence  by  passage 
through  guinea-pigs  and  chickens  produces  a  fatal  result.  Dogs,  sheep  and 
bovine  animals  succumb  to  intra-venous  inoculation. 

Pigs  as  a  rule  after  being  inoculated  sub-cutaneously  merely  suffer  from  a 
local  osdema  at  the  site  of  inoculation  and  a  transitory  rise  of  temperature  ; 
when  inoculated  however  with  a  very  virulent  virus  a  fatal  result  may  ensue. 

[*  Hog  cholera  is  also  known  as  swine  fever  and  swine  typhoid  and  in  Germany  as 
Schweinepest.  ] 


THE  SWINE  PASTEURELLA  455 

Post  mortem,  the  spleen  and  liver  are  enlarged,  there  are  patches  of  broncho - 
pneumonia  in  the  lungs,  and  pericarditis  ;  the  blood  is  dark-coloured  and 
like  pitch  :  the  organism  is  present  in  very  large  numbers  in  the  blood,  liver, 
spleen,  pericardial  fluid,  etc. 

Intra-venous  inoculation  leads  to  more  severe  symptoms  :  if  the  culture 
be  virulent,  death  from  septicaemia  soon  takes 

place:    with  an  attenuated  virus  a  condition  .'   ^  >::  *  •  • 

of   cachexia    accompanied    by   synovitis    and  *      *   /    ..'.'•*.' 

arthritis  is  more  or  less  rapidly  set  up.  "'•*.'„'      •  *  •  '  ,^y* 

It  is  difficult  to  infect  pigs  by  feeding  them.        •  • '  j»  .  •  /'•   ..    \  •  /  m~* 

Morphology. — Morphologically  the  organism  _ .  •  ..  -  •*"  '  i  /,  •.  "  • 
is  identical  with  the  other  members  of  the 
pasteurella  group.  The  swine  pasteurella  how- 
ever grows  more  easily  than  the  fowl  pasteurella ; 
it  can  be  grown  at  20°  C.  and  can  be  cultivated 
anaerobically  with  but  little  difficulty.  •:  '  *  •*  *  %- 

Vaccination.    Serum  therapy. — Swine  which  -      •*•       ^ .. 

have  recovered  from  an  attack  of  contagious 

pneumonia  are  found  to  be  immune.    Immunity      FIG.  zzs.—Pasteuretta  suis.    Film 
may  be   produced   experimentally  by  the   in-   (o^iv^obj^fth,  ReichT  C 
oculation  of  blood  sterilized  by  heat  (Selander 

and  others)  or  by  the  injection  of  small  doses  of  the  virus  (Metchnikoff)  or  of 
old  cultures  (Detmers). 

Rabbits  may  be  immunized  by  inoculating  them  with  the  serum  of  rabbits 
which  have  been  vaccinated  with  small  doses  of  the  virus  (Metchnikoff). 
De  Schweinitz,  Reters  and  Leclainche  have  also  obtained  a  prophylactic  and 
therapeutic  serum. 

Rabbits  which  have  been  vaccinated  with  attenuated  strains  of  the  swine  bacillus 
are  immunized  not  only  against  the  swine  bacillus  but  also  against  the  fowl  and 
rabbit  varieties.  Fowls  which  survive  the  inoculation  of  the  swine  bacillus  are 
immune  against  the  bacillus  of  fowl  cholera  (Chamberland  and  Jouan). 

Chamberland  and  Jouan  having  immunized  an  horse  against  the  swine 
bacillus  by  sub-cutaneous  inoculation  showed  that  the  serum  of  the  horse 
possessed  prophylactic  properties  equally  for  the  swine,  the  avian  and  the 
rabbit  bacillus.  It  agglutinated  most  strains  of  the  pasteurella  group,  viz.  : 
the  swine  bacillus  in  dilutions  of  1  in  60,000,  the  pasteurella  of  guinea-pigs 
in  1  in  4,000,  the  fowl  and  ovine  varieties  in  1  in  1,000. 


4.  PASTEURELLA  BOVIS. 

Under  the  name  "  Wild  und  Rinderseuche  "  Bellinger  first  described  an 
epizootic  disease  occurring  among  stags,  wild  boars,  deer,  roebucks  and  cattle, 
and  caused  by  an  ovoid  bacterium.  The  disease  sometimes  takes  the  form 
of  an  acute  haemorrhagic  septicaemia,  sometimes  it  is  more  chronic  and 
accompanied  by  pulmonary  localizations.  Oreste  and  Armanni  found  an 
identical  micro-organism  in  an  epizootic  disease  of  buffaloes — "  Barbone  " — and 
numerous  investigators  have  since  described  similar  epizootics  in  which  the 
same  organism  was  found  (Galtier,  Billings,  Smith,  Nocard,  Piot-Bey  and 
others).  In  the  Argentine,  Lignieres  observed  various  clinical  forms  (acute 
enteritis,  pleuro-pneumonia  and  haemorrhagic  septicaemia)  of  an  epizootic 
disease  caused  by  one  and  the  same  micro-organism  which  was  indistinguishable 
from  the  foregoing. 

These  various  affections  may  be  classed  together  under  the  general  term 


456  THE  PASTEURELLA  GROUP 

"  Bovine  pasteurelloses."  The  bovine  pasteurella  is  fatal  to  mice,  rabbits  and 
guinea-pigs  on  sub-cutaneous  inoculation,  and  to  oxen,  sheep,  dogs,  horses, 
pigs,  pigeons  and  fowls  on  intra- venous  inoculation.  The  cultural  charac- 
teristics and  staining  reactions  are  the  same  as  those  of  the  fowl  pasteurella. 
Similar  methods  must  also  be  adopted  in  examining  tissues  for  the  organism 
and  in  isolating  the  bacillus  ;  it  can  only  be  found  in  the  tissues  of  animals 
which  have  died  of  the  acute  forms  of  the  disease,  and  it  gives  very  scanty 
growths  under  anaerobic  conditions. 

Oreste  and  Armanni  have  been  able  to  vaccinate  buffaloes  against  barbone 
by  inoculating  them  with  attenuated  cultures. 

Vassal  immunized  a  number  of  calves  by  introducing  into  the  peritoneal 
cavities  of  the  animals  a  Chamberland  bougie  filled  with  a  broth  culture  of 
the  bacillus  and  hermetically  sealed.  Animals  thus  immunized  with  the 
toxin  stood  the  test  inoculation  and  also  yielded  a  serum  having  therapeutic 
properties. 

5.  PASTEURELLA  OVIS. 

Different  names  have  been  given  to  the  epizootic  disease  of  sheep  caused 
by  the  sheep  pasteurella,  e.g.  pneumo-enteritis  (Galtier)  ;  various  epizootics 
described  by  Lignieres  and  perhaps  also  some  described  by  other  investigators 
(Mercanti  and  Deny,  Benoist  and  Caille  and  others)  are  due  to  this  bacillus. 

The  sheep  pasteurella  has  all  the  characteristics  of  the  group  as  already 
described.  It  is  somewhat  difficult  to  grow  when  taken  direct  from  animals 
which  have  died  of  the  spontaneous  disease.  The  bacillus  is  always  found  in 
the  acute  but  very  rarely  in  the  chronic  forms  of  the  disease  :  it  is  pathogenic 
for  mice,  rabbits,  guinea-pigs,  dogs,  sheep  and  oxen. 


6.  PASTEURELLA  CAPR^E. 
(The  bacillus  of  infectious  pneumonia  of  goats.) 

The  infectious  pneumonia  of  goats  which  occurs  at  the  Cape,  in  India, 
Germany,  France,  Turkey  and  elsewhere  has  been  investigated  bacterio- 
logically  by  M.  Nicolle  and  Refik  Bey  in  Turkey.  It  is  caused  by  a  Pasteurella 
which  is  easily  isolated  from  the  lesions  in  the  lungs  and  from  the  mucous 
secretions. 

This  micro-organism  has  all  the  group  characters  of  the  pasteurella.  It  is 
rapidly  fatal  to  mice,  rabbits  and  pigeons  on  sub-cutaneous  inoculation,  to 
guinea-pigs  when  inoculated  intra-peritoneally,  and  to  goats  and  calves  on 
intra-pulmonary  inoculation.  Sub-cutaneous  inoculation  of  goats  slowly 
produces  a  condition  of  cachexia. 

Animals  can  be  immunized  by  the  inoculation  of  sterilized  cultures. 


7.  PASTEURELLA  EQUI. 

(The  bacillus  of  haemorrhagic  septicaemia  of  horses.) 

A  large  number  of  diseases  of  horses  having  very  different  clinical  features 
(typhoid  fever,  influenza,  contagious  pneumonia,  pneumo-enteritis,  pernicious 
anaemia)  appear  to  be  due  to  one  and  the  same  micro-organism,  the  equine 
pasteurella  (Lignieres)  ;  but  the  primary  (pasteurella)  infection  may  be 
followed  by  a  secondary  infection,  so  that  the  former  may  become  obscured. 
Possibly  the  disease  itself  is  a  secondary  infection  following  an  infection 
with  an  invisible  micro-organism  (Chap.  LXIV.). 


THE   CANINE   PASTEURELLA  457 

The  equine  pasteurella  often  passes  unnoticed  while  the  micro-organisms 
of  the  secondary  infection — especially  in  cases  of  strangles  and  contagious 
pneumonia — are  found  in  large  numbers. 

Cultures  are  only  obtainable  with    difficulty   direct  from  the  tissues   of 
the    horse.     To    recover    the    organism    from 
suspected    material  it  is  best   to   inoculate    a  I 

guinea-pig  intra-peritoneally  and  then  to  sow  .-•.+  f    ,     \     „" 

cultures  with  the  peritoneal  fluid  of  the  guinea-        f  ~ 
pig,  but  even   then   it   is  often  impossible  to      ^  .     V** 

recover  the  organism.  :/          "    *      1* ,  • 

The    equine    bacillus    kills    guinea-pigs    and     uv^V--* 
rabbits  and  sometimes  horses  on  sub-cutaneous        *  •    •  •    .  • 
inoculation.     Horses   succumb  to  intra-venous       .•.-,•'*'  Z ~  . 

inoculation.     It  is  only  slightly  pathogenic  for  ^  *  ^ 

fowls  and  pigeons  and  these  birds  only  succumb  V-.'  * .-  +. 

after  intra-venous  inoculation  of  one  or  several 

cubic  centimetres  of  the  peritoneal  exudate  of  Fm  ^.-Pa^ureiu  equi.  Film 
an  infected  guinea-pig.  Rats  and  oxen  are  from  the  peritoneal  exudate  of  a 
immune  guinea-pig— carbol-thionin. 

Morphologically  the  organism  differs  in  no  way  from  the  other  members 
of  the  group. 

8.  PASTEURELLA  CAN  IS. 

(The  bacillus  of  distemper.) 

Distemper,  which  attacks  chiefly  young  animals  and  assumes  very  various 
forms  (typhoid  fever,  dog-plague,  dog-pox,  infectious  pneumonia,  gastro- 
enteritis etc.),  [has  been  attributed]  to  a  pasteurella  (Lignieres,  Phisalix). 
Distemper  in  the  cat  is  due  to  the  same  organism  (Lignieres). 

Phisalix  investigated  a  septicsemic  condition  in  guinea-pigs  accompanied  by  the 
lesions  of  pneumonia.  He  found  it  to  be  due  to  a  small  bacillus  having  all  the 
morphological  and  cultural  features  of  a  typical  pasteurella  and  pathogenic  for 
rabbits,  mice,  pigeons  and  dogs.  Phisalix  considered  that  his  bacillus  was  identical 
with  that  which  Lignieres  later  described  in  distemper  in  dogs. 

The  micro-organism  of  distemper  is  somewhat  difficult  to  isolate  from  the 
tissues  of  the  dog  :  it  can  only  be  found  in  the  acute  forms,  and  is  most 
easily  recovered  from  the  blood  stream,  but  secondary  infections  rapidly 
supervene  with  the  result  that  the  pasteurella  disappears. 

Carre  diluted  the  nasal  secretions  with  sterile  water  and  after  filtering  the 
emulsion  through  a  very  porous  bougie  obtained  a  filtrate,  sterile  on  cultiva- 
tion but  capable  of  infecting  fresh  animals.  From  these  experiments  it  may 
be  concluded  that  the  true  virus  of  distemper  is  an  invisible  micro-organism, 
the  pasteurella  being  present  merely  as  a  secondary  infection.  It  may  be 
necessary  to  review  the  aetiology  of  "  distemper,"  since  it  is  possible  that 
some  of  the  clinical  conditions  known  by  this  name  are  due  to  a  pasteurella 
others  to  a  filtrable  virus  (Chap.  LXIV.)  [and  see  also  M'Gowan's  bacillus, 
infra]. 

Experimental  inoculation. — The  dog  pasteurella  is  only  slightly  virulent 
for  animals  other  than  the  dog  and  cat. 

It  kills  mice  and  guinea-pigs  on  intra-peritoneal  inoculation  with  the 
lesions  of  acute  peritonitis.  Post  mortem,  the  intestine,  liver,  kidneys  and 
spleen  are  congested  :  the  organism  can  be  recovered  from  the  blood  and  is 
also  present  in  large  numbers  in  the  peritoneal  exudate. 

Rabbits  succumb  to  intra-peritoneal  inoculation  :  intra-venous  and  sub- 
cutaneous inoculation  is  also  fatal  if  a  very  large  dose  of  the  virus  be  given, 


458  THE   PASTEURELLA  GROUP 

or  if  a  bacillus  the  virulence  of  which  has  been  increased  by  passage  through 
rabbits  or  guinea-pigs  be  inoculated.  Birds  (fowls,  pigeons  and  ducks)  can 
only  be  killed  by  inoculation  of  a  bacillus  the  virulence  of  which  has  been 
increased  by  passage  through  guinea-pigs. 

Broth  cultures  of  a  strain  recently  isolated  from  the  tissues  of  a  dog 
are  pathogenic  for  dogs  and  cats.  Sub-cutaneous  inoculation  of  such  cultures 
produces  a  disease  which  in  adult  dogs  tends  to  recovery  but  which  in  young 
dogs  is  often  fatal.  When  the  animal  dies  within  4  or  5  days  of  the  inocula- 
tion the  micro-organism  can  be  found  in  the  blood,  internal  organs,  glands, 
and  in  the  oedema  at  the  site  of  inoculation.  If  death  does  not  take  place 
until  after  the  lapse  of  5  or  6  days,  cultures  sown  with  the  blood  or  scrapings 
from  the  internal  organs  yield  various  organisms  but  not  the  pasteurella  ; 
the  latter  may  however  be  found  in  the  fluid  from  the  local  oedema  at  the 
site  of  inoculation  and  in  the  lymphatic  glands. 

Intra-venous  inoculation  of  cultures  is  fatal  to  dogs  and  gives  rise  to  the 
various  clinical  symptoms  of  the  disease.  Inoculation  with  small  doses  of 
the  virus  produces  gastro-intestinal  symptoms.  The  condition  though 
apparently  tending  to  recovery  slowly  leads  to  death  from  cachexia ;  the  micro- 
organism can  only  be  isolated  from  the  tissues  during  the  first  week  after 
inoculation.  Lignieres  failed  to  infect  dogs  by  feeding  them. 

It  is  very  difficult  to  transmit  the  disease  by  direct  inoculation  of  material 
obtained  from  diseased  animals  :  the  fluid  in  the  pustules  is  not  virulent. 
Occasionally,  when  the  discharge  from  the  nose  or  the  pulmonary  exudate 
or  blood  have  been  used,  sub-cutaneous  or  intra-venous  inoculation  has 
given  positive  results.  The  most  successful  results  have  been  obtained  by 
painting  the  nasal  fossae  of  young  dogs  with  the  nasal  discharge  of  sick 
animals. 

Morphology. — The  canine  pasteurella  exhibits  the  ordinary  morphological 
features  of  the  group  :  when  taken  direct  from  the  tissues  of  the  dog  it  is  a 
fairly  long  bacillus,  but  after  the  first  passage  through  guinea-pigs  it  assumes 
the  shape  of  a  cocco-bacillus.  It  grows  well  at  18°-20°  C.  and  better  on 
coagulated  serum  than  on  agar. 

Toxin. — Phisalix  obtained  a  toxin,  which  killed  rabbits,  by  growing  the 
canine  pasteurella  in*  ordinary  broth  or  better  in  peptonized  Liebig's  broth 
for  5  days,  and  with  it  was  able  to  reproduce  experimentally  most  of  the 
clinical  features  of  the  disease  as  seen  in  dogs  :  it  lowers  the  resistance  of  the 
inoculated  animal  and  favours  the  development  of  secondary  infections. 
Guinea-pigs  are  only  slightly  susceptible  to  the  action  of  the  toxin. 

The  toxin  obtained  on  killing  cultures  with  ether  or  chloroform  is  found 
to  be  much  more  virulent  than  that  prepared  by  filtration. 

Vaccination. — Phisalix,  applying  the  method  of  attenuation  adopted  by 
Pasteur  for  fowl  cholera,  has  prepared  a  vaccine  which  seems  to  give  good 
results  in  veterinary  practice. 

Dogs  should  be  vaccinated  when  about  2  months  old.  Two  inoculations 
(each  of  about  3  c.c.)  are  given  beneath  the  skin  at  an  interval  of  a  fortnight. 
The  inoculation  is  not  dangerous  and  immunizes  the  dog  sufficiently  to  protect 
it  against  the  spontaneous  disease. 

The  vaccines  of  Phisalix  do  not  protect  dogs  against  intra-venous  inoculation. 
For  practical  purposes  there  is  no  need  to  produce  a  high  degree  of  immunization, 
and  moreover  if  carried  too  far  it  may  lead  to  the  development  of  visceral  and  more 
particularly  renal  lesions  (dogs  can  be  highly  immunized  by  repeatedly  inoculating 
them  with  viruses  of  increasing  virulence). 

Lignieres  prefers  to  use  a  polyvalent  vaccine  which  gives  more  constant 
results  than  the  monovalent  vaccine  of  Phisalix. 


IMMUNIZATION  OF  ANIMALS  459 

[M'Gowan's  bacillus  of  distemper.] 

[M'Gowan1  lias  recently  brought  forward  evidence  to  show  that  "  Dis- 
temper "  is  due  to  a  gram-negative,  non-spore-bearing  slightly  motile  bacillus. 

[  The  organism  was  recovered  from  a  large  number  of  more  or  less  diseased 
animals  of  different  species  :  in  all  the  primary  focus  of  the  disease  was  the 
respiratory  tract.  The  dogs  and  cats  from  which  the  organism  was  isolated 
showed  the  symptoms  commonly  associated  with  "  distemper." 

[The  organism  measures  when  taken  from  the  tissues  from  0'5-2'3/x  long  by 
about  0'4-0'5/A  broad.  It  grows  on  all  the  ordinary  media  both  aerobically 
and  anaerobically  and  does  not  liquefy  gelatin. 

[  It  forms  neither  acid  nor  gas  in  a  peptone-salt  medium  containing  any  of 
the  following  carbohydrates  :  lactose,  saccharose,  salicin,  mannite,  dulcite, 
maltose,  galactose,  raffinose,  glucose,  inulin,  inosite,  adonite.  On  the  other 
hand,  the  solutions  become  markedly  alkaline  after  a  few  days. 

[Litmus  milk  is  turned  alkaline  and  not  coagulated. 

"  On  potato  the  growth  is  characteristic,  the  appearance  being  that  of  a 
buff-coloured,  or  yellow  to  copper-brown,  raised,  moist,  heavy  growth.  The 
brownish  colour  is  perceptible  in  24  hours  and  is  very  evident  in  48  hours  " 
(M'Gowan). 

[On  intra-peritoneal  inoculation  the  organism  was  pathogenic  to  a  large 
variety  of  animal  species  including  dogs  and  cats,  and  there  is  evidence  "  that 
the  organism  in  pure  culture  can  produce  in  healthy  dogs,  when  applied  to 
their  nasal  mucous  membrane,  the  clinical  symptoms  of  "distemper."] 


9.  IMMUNIZATION  WITH  THE  POLYVALENT  VACCINES 
OF  LIGNIERES. 

Starting  with  the  idea  that  an  animal  may  be  infected  with  several  varieties  of 
pasteurella,  Lignieres  recommends  the  use  of  polyvalent  vaccines.  The  vaccine 
is  prepared  by  mixing  cultures  of  the  sheep,  ox,  dog,  horse,  pig,  and  fowl  varieties. 
In  order  to  avoid  any  changes  in  virulence,  only  cultures  which  have  been  grown  on 
agar  in  the  laboratory  for  at  least  a  year  and  have  been  re-sown  every  other  day 
are  used.  To  prepare  the  vaccines,  these  cultures  are  sown  in  flat- bottomed  flasks 
containing  a  shallow  layer  of  broth  and  incubated  at  42°-43°  C.  for  5  days  for  the 
first  vaccine  and  for  2  days  for  the  second. 

The  dose  of  each  vaccine  varies  from  O'125-l  c.c.  according  to  the  size  of  the 
animal,  and  the  two  inoculations  are  given  sub-cutaneously  at  intervals  of  12  days 
or  a  fortnight.  The  resulting  immunity  lasts,  on  an  average,  12  months. 

Polyvalent  serum. 

Lignieres  and  Spitz  prepared  a  polyvalent  serum  which  was  both  prophylactic 
and  curative. 

Mixed  cultures  of  the  six  varieties  of  the  pasteurella  which  have  been  kept  on 
agar  for  a  year,  and  sown  as  described  above,  are  injected  into  horses  in  repeated 
small  doses  (5-20  c.c.)  at  intervals  of  a  few  days  first  under  the  skin  then  into  the 
veins.  After  each  inoculation  the  animals  show  a  sharp  reaction  lasting  2  or  3  days. 

The  serum  so  obtained  is  prophylactic  and  curative  but  has  no  antitoxic  properties. 
In  doses  of  40-60  c.c.  it  gives  the  best  results  in  the  treatment  of  the  equine  pas- 
teurellosis,  and  in  doses  of  5-10  c.c. — if  given  at  the  onset  of  the  disease — it  is  [said 
to  be]  very  efficacious  in  the  treatment  of  distemper  in  dogs. 


Journal  of  Pathology  and  Bacteriology,  xv.  p.  372. 


CHAPTER  XXIX. 
BACILLUS  PESTIS. 

Introduction. 

Section  I. — The  experimental  disease,  p.  463. 

Section  II. — Morphology,  p.  464. 

1.  Microscopical  appearances  and  staining  reactions,  p.  464.  2.  Cultural  charac- 
teristics, p.  465. 

Section  III. — Biological  properties,  p.  466. 

1.  Vitality  and  virulence,  p.  466.  2.  Bio-chemical  reactions,  p.  467.  3  Toxins, 
p.  467.  4  Vaccination,  p.  468.  5.  Serum  therapy,  p.  471.  6.  Agglutination, 
p.  472.  7.  Precipitins,  p.  473. 

Section  IV. — Isolation  and  identification  of  the  plague  bacillus  (including  an  account  of 
the  post  mortem  appearances  in  the  naturally  infected  rat),  p.  473. 

THE  bacillus  of  plague  was  discovered  in  1894  by  Yersin  [and  Kitasato 
independently]. 

In  the  human  subject  plague  may  assume  one  of  two  forms,  bubonic  or  pneumonic  ; 
of  the  two  the  former  is  the  more  common.  [In  both  forms  a  septicaemia  may  occur, 
generally  as  a  late  symptom,  but  it  is  incorrect  to  speak  of  a  septicsemic  as  opposed 
to  a  bubonic  and  a  pneumonic  form  (Simond).] 

In  bubonic  plague  the  bacillus  is  present  in  the  pus  of  the  lymphatic  glands  and 
occasionally  in  the  blood  and  more  rarely  in  the  stools  (Wilm).  [In  India  the 
Advisory  Committee  x  found  that  in  a  large  proportion  of  cases  (67  per  cent.)  the 
bacillus  is  present  in  the  blood  and  that  a  bacilluria  occurs  in  about  30  per  cent,  of 
cases.  In  bubonic  plague  it  is  not  uncommon  for  a  secondary  pneumonia  to 
develop.] 

In  the  pneumonic  form,  though  there  is  an  absence  of  buboes,  the  bacillus  is 
present  in  the  lymphatic  glands.  It  is  frequently  present  in  the  blood  and  always 
in  the  sputum  (Metin  indeed  demonstrated  the  presence  of  bacilli  in  the  sputum  of 
plague  patients  a  week  after  the  temperature  had  fallen  to  normal,  but  could  detect 
them  only  by  inoculation  and  found  their  virulence  was  attenuated)  ;  bacilli  can 
also  be  found  in  the  juice  and  in  sections  of  the  lung  and  spleen  (Tchistowitch). 

According  to  Haffkine  the  native  races  of  India  are  more  susceptible  to  plague 
than  are  either  white  people  or  the  native  races  of  Africa. 

In  epidemics  of  plague,  rats  are,  as  a  rule,  the  first  to  suffer.  "  Plague 
which  is  at  first  a  disease  of  rats  soon  becomes  a  disease  of  man  "  (Roux  and 
Yersin). 

[Besides  its  occurrence  in  rats  natural  plague  has  been  observed  in  guinea-pigs 
in  India  (Indian  Commission);  in  rabbits  in  India  (Indian -Commission)  and  in 
England  (Martin  and  Rowland)  ;  in  apes — Cynopithecus  niger — and  monkeys — 

[*  The  reports  on  plague  investigations  in  India  issued  by  the  Advisory  Committee 
appointed  by  the  Secretary  of  State  for  India,  the  Royal  Society  and  the  Lister  Institute 
are  published  in  the  Journal  of  Hygiene,  1906,  1907,  1908  and  1910.] 


PLAGUE  461 

Macacus  sinensis,  Semnopithecus  entellus,  Macacus  nemestrinus — in  the  Bombay 
Zoological  Gardens  (Indian  Commission) ;  in  cats  in  the  Azores  (de  Souza,  Arruda 
and  Pinto) ;  in  ground-squirrels — Citellus  beccheyi — in  America  (M'Coy)  ;  and  in 
brush  rats — Neotoma — (M'Coy).  The  disease  is  also  believed  to  occur  in  the  mar- 
mot— Arctomys  bobax — (vide  infra).  The  infection  of  the  ground-squirrels  on  the 
Pacific  Coast  of  the  United  States  of  America  was  believed  to  be  the  source  of 
infection  in  a  number  of  cases  of  plague  in  that  district.  ] 

During  epidemics  of  plague  a  large  number  of  animals  [other  than  those  men- 
tioned have  been  said  to  be]  affected  at  the  same  time  as  man  :  [Simpson,  for 
example,  stated  that  pigs,  calves,  buffaloes,  sheep,  hens,  ducks,  geese,  turkeys 
and  pigeons  are  susceptible.  But  Bannerman  and  Kapadia,  as  Members  of  the 
Commission  appointed  by  the  Advisory  Committee,  failed  to  infect  pigs,  calves, 
fowls,  turkeys,  geese  and  ducks,  and  showed  that  buffaloes  are  not  susceptible. 
The  conclusions  of  the  Indian  Commission  are  supported  by  many  other  observers 
among  whom  may  be  mentioned  Pearse  in  Hong-Kong,  London  in  Russia,  Watkins- 
Pitchford  in  Natal  and  de  Souza,  Arruda  and  Pinto  in  the  Azores.  The  last  named 
observers  further  showed  that  dogs  are  practically  refractory  to  plague  and  that 
ferrets  are  only  susceptible  to  large  doses  of  the  virus.  From  these  and  other  facts  it 
appears,  as  Bannerman  points  out,  that  Simpson  confused  the  plague  bacillus  with 
the  hog-cholera  bacillus  (p.  438)  and  with  the  bacillus  of  fowl  cholera  (p.  447).] 

Plague  is  transmitted  from  rat  to  rat  and  from  rat  to  man  by  fleas  (Simond). 
[The  rat  flea,  Xenopsyllus  (Pulex )  cheopis  Rothschild,  under  certain  circumstances 
is  attracted  by  man  and  will  readily  bite  and  feed  on  him  (Advisory  Com- 
mittee).1] 

[It  is  possible  to  transmit  plague  by  means  of  Pulex  irritans.  Nevertheless  the 
direct  transmission  of  the. disease  from  man  to  man  cannot,  at  the  present  time,  be 
of  frequent  occurrence  or  we  should  have  evidence  of  direct  infection  instead  of 
dependence  upon  the  epizootic.  The  reason  why  the  human  flea  is  ineffective  is 
because  in  human  cases  the  average  degree  of  septicaemia  before  death  is  so  much 
less  than  in  rats  that  the  chance  of  a  flea  imbibing  even  a  single  bacillus  is  small 
(C.  J.  Martin).] 

Rats  suffering  from  plague  are  a  ready  prey  to  fleas  and  plague  bacilli  have  been 
found  in  the  stomachs  of  these  insects.  If  a  flea  be  taken  from  a  plague-infected 
rat,  crushed  in  a  mortar  and  inoculated  into  a  mouse,  the  latter  becomes  infected 
with  plague  [Ogata]. 

[There  is  good  evidence  that  the  plague  bacillus  multiplies  in  the  stomachs  of 
fleas  (Indian  Commission).] 

[Simond  holds  that  when  a  flea  bites  man  or  the  lower  animals  it  discharges  the 
contents  of  its  intestine  near  the  bite  ;  should  the  flea  be  infected  the  plague  bacillus 
— which  will  be  present  in  the  excreta — will  be  rubbed  into  the  bite  by  the  scratching 
induced  as  a  result  of  the  bite.  The  experiments  of  the  Advisory  Committee  would 
appear  to  support  this  view.  Both  male  and  female  fleas  bite.] 

A  healthy  rat  coming  in  contact  with  fleas  from  a  plague-infected  rat  dies  of 
plague.  When  a  rat  dies  the  fleas  leave  the  carcase. 

Plague  is  similarly  transmitted  by  fleas  from  man  to  man.  In  the  early  stages 
of  an  infection  with  the  plague  bacillus  small  inflamed  areas  are  occasionally  seen, 
varying  in  size  from  a  pin's  head  to  a  walnut,  transparent  at  first  then  purulent  and 
always  containing  bacilli.  These  inflammations  are  found  on  parts  exposed  to  the 
bites  of  insects,  and  it  would  appear  that  they  mark  the  sites  of  inoculation.  Sticker, 
working  in  Bombay,  pricked  himself  with  an  infected  instrument  and  after  3  days 
an  inflammation  appeared  at  the  site  of  the  injury  and  symptoms  of  plague  manifested 
themselves. 

[*  Xenopsyllus  cheopis  is,  except  in  Northern  and  Central  Europe,  the  commonest  flea 
found  on  house  and  port  rats  all  over  the  world  and  in  some  localities  is  almost  the  only 
flea  found.  Ceratophyllus  fasciatus  is  the  flea  usually  found  on  Mus  decumanus  in  Great 
Britain  (M.  rattus  is  a  rare  animal  in  the  British  Islands),  and  this  is  also  the  case,  apparently, 
throughout  Northern  and  Central  Europe  (Rothschild). 

[X.  cheopis  is  identical  with  P.  pallidus  Taschenberg,  with  P.  murinus  Tiraboschi 
and  with  P.  philippinensis  Hertzog. 

[P.  irritans,  the  flea  commonly  found  on  man,  Ctenocephalus  canis,  of  dogs,  cats,  etc.,  and 
Ctenopsyllus  musculi,  the  common  house  mouse  flea,  have  occasionally  been  found  on 
rats.] 


462  THE   PLAGUE   BACILLUS 

[Plague  may  also  possibly  be  transmitted  from  man  to  man  through  the  agency  of 
bugs.  Nuttall  found  plague  bacilli  in  the  intestinal  canal  of  bugs  and  has  shown  that 
they  can  convey  the  infection  of  plague  from  infected  to  healthy  animals.  Verjbitski 
showed  that  the  ordinary  domestic  bug,  Cimex  lectularius,  will  bite  mice,  rats,  and 
guinea-pigs,  and  that  plague  bacilli  can  be  recovered  from  these  insects  for  periods 
varying  from  1-8  days  after  they  have  been  fed  on  septicsemic  animals.  Jordansky 
and  Kladnitsky  find  that  the  plague  bacillus  retains  its  virulence  in  the  bodies  of 
bugs  for  10  days  and  more.] 

Flies  may  also  play  a  part  in  the  dissemination  of  plague.  They  die  in  large 
numbers  during  an  epidemic  of  plague  and  the  bacillus  can  be  found  in  their  bodies 
(Yersin).  [Jordansky  and  Kladnitsky  are  however  of  opinion  that  neither  flies, 
cockroaches  nor  ants  play  an  important  part  in  plague. 

[Ogata  has  suggested  that  mosquitoes  may  convey  the  infection  from  the  diseased 
to  the  healthy  subject.] 

Infection  by  feeding. — Man  only  very  rarely  becomes  infected  through  the  ali- 
mentary canal  (Wilm  has  recorded  one  case  in  which  the  most  prominent  symptoms 
were  intestinal  and  in  which  the  bubo  was  found  to  be  in  the  mesenteric  glands). 
[With  regard  to  animals  the  Advisory  Committee  find  that  "  in  nature  intestinal 
infection  rarely  or  never  takes  place  and  that  in  consequence  rats  do  not  become 
infected  by  eating  the  carcases  of  their  comrades "  ;  cf.  experimental  feeding 
experiments  (p.  464).] 

["  The  position  of  knowledge  on  the  question  of  the  importance  of  alimentary 
infection  in  the  spread  of  plague  may  be  summarized  as  follows : 

[1.  Contamination  of  aliments  may  conceivably  lead  to  the  infection  of  human 
beings  on  occasion,  but  the  chance  of  bacilli  reaching  foodstuffs  destined  for  con- 
sumption uncooked,  and  in  which  they  would  multiply  greatly,  are  slight. 

[2.  The  alimentary  canal  is  not  an  easy  method  of  infecting  animals,  large  quan- 
tities of  virulent  bacilli  being  usually  necessary. 

[3.  There  is  absolutely  no  epidemiological  evidence  pointing  to  alimentary  infec- 
tion being  anything  but  uncommon,  and  in  about  75  per  cent  of  human  cases, 
the  situation  of  buboes  indicates  skin  infection  "  (C.  J.  Martin).] 

Infection  by  the  respiratory  passages  is  easily  effected  in  animals,  and  is  not 
of  rare  occurrence  in  man  :  "it  would  appear  to  be  the  only  channel  of  infection 
in  pneumonic  cases  "  (Balzaroff).  ["  Pneumonic  plague  may  arise  by  the  inhalation 
of  bacilli  into  the  lungs,  where  they  rapidly  multiply  and  early  gain  access  to  the 
blood  stream,  or  bacilli  which  have  gained  entrance  through  any  other  channel 
and  have  become  generalized  may  subsequently  establish  themselves  in  the  lungs 
and  occasion  a  secondary  pneumonia.  Some  degree  of  secondary  pneumonia  is  not 
uncommon  in  man  and  animals  suffering  from  bubonic  plague.  A  case  of  bubonic 
plague  may  therefore  become  a  potential  source  of  a  pneumonic  outbreak.  The 
spread  of  the  pneumonic  form  of  the  disease  offers  no  difficulties,  since  it  was  shown 
by  Childe  that  the  sputum  of  these  cases  contains  innumerable  bacilli,  and  by  Martini 
that  plague  pneumonia  is  readily  produced  in  animals  exposed  to  an  atmosphere 
containing  droplets  of  an  emulsion  of  plague  culture.  Pneumonic  plague  is  obviously 
spread  by  man-to-man  infection  "  (C.  J.  Martin). 

[In  the  recent  epidemic  of  pneumonic  plague  in  Manchuria,  rats  were  not  attacked 
by  the  disease,  but  the  tarbagan  (Arctomys  bobax1)  was  held  to  be  responsible  for 
the  presence  of  endemic  plague  in  Mongolia  and  in  the  Russian  provinces  adjacent. 
The  connexion  between  the  tarbagan  and  plague  has  not  been  satisfactorily  worked 
out,  but  all  the  evidence  available  seems  to  show  that  there  might  be  truth  in  the 
belief  of  the  infective  power  of  the  marmot  in  this  connexion.  The  origin,  how- 
ever, of  the  pneumonic  plague  in  Manchuria  has  yet  to  be  discovered  (Petrie). 

[However  probable  it  may  seem  on  the  evidence  at  present  available  it  cannot 
be  said  to  be  proved  that  contact  alone  is  the  only  factor  concerned  in  the  spread 
of  pneumonic  plague.  Though  "  some  degree  of  secondary  pneumonia  is  not  un- 
common in  man  and  animals  suffering  from  plague  "  (Martin)  yet  the  experience 
in  plague  hospitals  in  India  is  opposed  to  the  view  that  infection  takes  place  by 
direct  contact  with  a  patient  suffering  from  plague  and  in  the  laboratory  the  Advisory 
Committee  found  that  "  fleas  and  fleas  alone  were  the  transmitting  agent  in  the 
experimental  production  of  plague  epidemics  among  animals."  Since  then  contact 

[JThe  Bobac  or  Polish  marmot.] 


EXPERIMENTAL  INFECTION  463 

can  be  definitely  excluded  as  a  source  of  infection  in  India  it  is  difficult  to  understand 
how  it  alone  will  explain  the  Manchurian  epidemic.] 

The  plague  bacillus  is  capable  of  retaining  its  vitality  outside  the  body.  Yersin 
recovered  a  plague  bacillus,  less  virulent  it  is  true  than  those  isolated  from  buboes, 
from  the  soil  of  an  infected  place.  [In  moist  earth  previously  sterilized  the  bacillus 
will  survive  for  months  (Gladin,  Marsh).]  In  the  bodies  of  dead  rats  the  organism 
can  retain  its  virulence  for  several  weeks  (Maassen).  According  to  Inghilleri  the 
plague  bacillus  is  able  to  live  in  drinking  water  for  about  a  month. 

SECTION  I.— THE   EXPERIMENTAL  DISEASE. 

Monkeys,  mice,  rats,  guinea-pigs  and  rabbits  are  all  very  susceptible  to 
experimental  infection  with  plague  :  [but  domestic  animals  such  as  horses, 
cattle,  sheep,  goats,  pigs  and  calves,  pigeons,  geese,  fowls,  ducks  and  turkeys 
are  apparently  not  susceptible  "  either  by  ingestion,  scarification  or  sub- 
cutaneous inoculation "  (Bannerman  and  Kapadia,  Haffldne,  London, 
Watkins-Pitchford,  de  Souza,  Arruda  and  Pinto).  ]  De  Mattei  affirms  however 
that  pigeons,  fowls  and  ducks  succumb  if  inoculated  with  large  doses  of 
virulent  cultures. 

Cultures  of  the  plague  bacillus  are  very  virulent  for  man,  and  accidents 
in  laboratories  have  shown  that  a  certain  element  of  danger  attaches  to 
working  with  the  organism. 

1.  Sub-cutaneous  inoculation. — To  infect  a  monkey,  a  mouse,   [or  other 
susceptible  animal]  with  plague  it  is  only  necessary  to  scratch  the  skin 
lightly  with  a  needle  charged  with  the  virus.     Rats  and  mice  die  in  2  or  3 
days,  guinea-pigs  in  2-5  days  and  rabbits  in  3-8  days. 

A  few  hours  after  inoculating  a  guinea-pig  a  localized  oedema  makes  its  appearance 
at  the  site  of  inoculation  followed  by  a  swelling  of  the  related  glands ;  at  the 
end  of  24  hours  the  animal  will  be  found  lying  on  its  side  and  its  coat  ruffled  : 
death  is  preceded  by  convulsive  seizures. 

Post  mortem  there  is  a  reddish  oedema  at  the  site  of  inoculation  and  around  the 
neighbouring  gland  :  the  abdominal  organs  are  congested  while  the  spleen  is  very 
much  enlarged  and  often  exhibits  an  eruption  simulating  small  miliary  tubercles. 
[When  the  disease  has  been  of  some  duration  abscesses  are  occasionally  found  in  the 
abdominal  wall.]  There  is  a  small  amount  of  serous  exudate  in  the  pleurae  and 
peritoneum  and  bacilli  can  be  demonstrated  in  the  fluid  :  bacilli  are  also  to  be  found 
in  large  numbers  in  the  lymphatic  glands,  liver,  spleen  and  blood. 

The  virulence  of  the  organism  is  increased  by  passage  through  guinea-pigs 
using  in  the  first  instance  a  scraping  from  the  spleen  or  a  little  blood. 

By  passing  the  virus  through  a  series  of  animals  of  the  same  species  bacilli  can 
be  obtained  which  are  of  an  exalted  and  constant  virulence  for  that  species.  For 
instance  a  bacillus  can  be  recovered  which  will  consistently  kill  a  mouse  in  2  days  ; 
one  which  will  kill  a  guinea-pig  in  2  or  3  days  ;  or  one  which  is  fatal  to  a  rabbit 
in  3  days.  A  bacillus  which  will  kill  a  mouse  in  2  days  takes  rather  a  long  time  to 
kill  a  rabbit,  but  after  being  passed  through  a  few  rabbits  will  kill  these  animals  in 
3  days  ;  it  has  however  now  lost  its  virulence  for  the  mouse  and  to  restore  its  viru- 
lence for  the  latter  species  it  must  be  passed  from  mouse  to  mouse  a  few  times 
(Yersin,  Calmette  and  Borrel). 

[No  alteration  in  virulence  for  rats  is  observed  after  sub-cutaneous  passage 
through  rats.] 

2.  Cutaneous  inoculation. — Guinea-pigs  are  readily  infected  by  rubbing 
infected  material  on  the  surface  of  the  shaved  skin  (Weichselbaum,  Albrecht 
and  Ghon).     A  slight  inflammation  first  forms  and  the  disease  then  runs  the 
same  course  as  in  the  previous  case. 

This  affords  a  valuable  test  for  the  detection  of  plague  bacilli  in  material  con- 
taminated with  other  organisms,  and  is  the  method  which  should  be  adopted  for 
the  detection  .of  the  bacillus  in  decomposing  carcases,  fecal  matter,  etc. 


464  THE   PLAGUE   BACILLUS 

3.  Intra- venous  inoculation. — Inoculation  into  the  veins  leads  to  a  more 
severe  disease  in  laboratory  animals  than  sub-cutaneous  inoculation :    apart 
from  the  local  lesion  the  symptoms  are  similar  in  the  two  cases. 

4.  Intra-peritoneal  inoculation. — This  mode  of  infection  is  very  severe  ;   an 
inoculated  guinea-pig  will  die  in  24-40  hours.     The  virulence  of  the  bacillus 
can  be  increased  by  passage  through  animals  by  means  of  collodion  sacs 
inserted  into  the  peritoneal  cavity  (Roux). 

5.  Infection  of  the  mucous  membranes. — An  animal  can  be  infected  through 
any  of  the  mucous  membranes  (nasal,  conjunctival,  buccal,  vaginal,  etc.). 

Rats,  mice,  guinea-pigs  and  rabbits  die  of  plague  if  a  trace  of  the  virus  be 
placed  on  the  nasal  mucous  membrane  without  injuring  it.  The  disease  can 
be  more  surely  transmitted  by  this  method  than  by  sub-cutaneous  inoculation 
(Roux  and  Balzaroff). 

An  attenuated  virus  which  fails  to  give  rise  to  a  fatal  infection  when  inoculated 
hypodermic  ally  will  produce  the  pneumonic  form  of  the  disease  when  inoculated  into 
the  respiratory  passages,  and  the  virulence  of  the  organism  can  be  recovered  in  this 
way,  for  example  by  successive  passages  on  the  nasal  mucous  membrane.  A  virus 
which  has  been  dried  even  for  several  weeks  in  albuminoid  matter  gives  rise  to  the 
pneumonic  form  of  the  disease  when  inoculated  into  the  nose. 

6.  Ingestion. — Rats,  mice  and  monkeys  may  be  infected  by  feeding  them 
on  living  cultures  (Simond)  or  on  the  viscera  of  plague-infected  animals. 

[The  Advisory  Committee  found  that  about  one-fourth  (26' 2  per  cent.) 
of  the  rats  which  they  fed  on  the  whole  carcases  or  viscera  of  infected  rats 
and  guinea-pigs  contracted  plague.  The  majority  died  on  the  third  and 
fourth  days  after  receiving  the  plague-infected  meat,  though  in  a  few  cases 
death  was  delayed  as  long  as  three  weeks.  Among  the  (437)  rats  which  were 
still  alive  at  the  end  of  three  weeks  11  showed  undoubted  signs  of  being 
plague-infected.  Post  mortem  examination  of  the  rats  which  died  showed 
lesions  similar  to  those  found  in  rats  naturally  infected  save  in  two  very 
important  particulars.  In  rats  infected  by  feeding  the  mesentery  was  by 
far  the  commonest  situation  for  the  bubo,  and  in  about  one-third  of  the 
number  the  Peyer's  patches  were  enlarged,  congested,  hsemorrhagic  and 
often  ulcerated,  and  the  intestines  markedly  congested  (cf.  appearances 
presented  by  naturally  infected  rats  p.  474).] 

7.  Contagion. — If  a  number  of  healthy  mice  be  placed  in  a  bottle  with  a 
number  of  inoculated  mice  the  former  contract  the  disease  and  die  with 
lesions   characteristic   of  plague   (Yersin)  :     [but   the   Advisory   Committee 

showed  that  "  close  contact  of  plague-infected 

t   0    +  animals  with  healthy  animals,  if  fleas  are  ex- 

0  -  ""  Q  4,  eluded,  does  not  give  rise  to  an  epizootic  among 

0      K^  9  **  ^    J^  the  latter  "  and  that  aerial  infection  is  not  a 

&  <*  *»    ^~f%#    '°  ^       means  whereby  the  disease  is  spread  from  an 

*    A**  Q  **  9    **       "  infected  animal  to  a  healthy  animal. ] 

<Q  *  ^**       &        ** 

"     -tr"  t*-« 

SECTION  II.— MORPHOLOGY. 
ff^?*  !•  Microscopical  appearance. 

g   * .  ?  The  micro-organism  of  plague  as  seen  in 

$  preparations  from  the  tissues  is  a  short,  squat 

bacillus  with  rounded  ends  and  is  more 
correc%  described  as  a  cocco-bacillus :  it 
measures  about  2xl/^.  [Morphologically 
it  very  closely  resembles  the  bacilli  of  the  pasteurella  group,  the  bacillus 
pseudo-tuberculosis  rodentium,  and  often  bacilli  of  the  typhoid-Qolon  group.} 


MORPHOLOGY  465 

The  bacillus  does  not  form  spores,  and  is  generally  said  to  be  non-motile, 
[but  Gordon  states  that  it  is  furnished  with  flagella  and  is  motile].  In  pre- 
parations made  from  blood  the  organism  is  rather  longer  than  in  the  buboes 
and  often  appears  as  though  surrounded  by  an  hyaline  capsule. 

In  broth  cultures  the  organism  grows  in  chains.  On  agar  more  or  less 
elongated  forms  are  seen  among  the  ordinary  short  cocco-bacillary  forms. 

In  old  cultures  and  on  agar  containing  salt  [2-5  per  cent.]  the  plague  bacillus  gives 
rise  to  involution  forms  consisting  of  large  ball- like  swellings  and  under  these 
conditions  the  organisms  stain  feebly  (fig.  232). 


FIG.  231. — Plague  bacillus.     From  a  broth          FIG.  232. — Plague  bacillus.    Involution  forms, 
culture.    (After  Yersin.)  Agar  cultures  (6  days).     ^Vn. 

Staining  methods. — The  plague  bacillus  stains  readily  with  the  ordinary 
basic  aniline  dyes  ;  carbol- violet  or  carbol-thionin  can  be  recommended.  It 
is  gram-negative. 

When  stained  with  weak  dyes  the  ends  of  the  organism  stain  more  deeply 
than  the  centre,  so  that  the  bacillus  often  presents  the  appearance  of  a 
shuttle. 

[To  obtain  good  polar-stained  bacilli  fix  the  film  by  heat,  float  the  cover- 
glass  on  to  the  staining  bath  (frichsin  1  per  cent.,  carbolic  acid  3  per  cent., 
glycerin  40  per  cent.),  wash  almost  immediately  in  60  per  cent,  alcohol,  pass 
rapidly  through  water  (Jordansky  and  Kladnitsky).  Giemsa's  and  Roma- 
nowsky's  stains  also  give  very  good  results.] 

2.  Cultural  characteristics. 

1.  Conditions  of  growth. — The  plague  bacillus  is  an  aerobic  organism.     It 
grows  easily  on  the  ordinary,  slightly  alkaline,  media.     Growth  begins  about 
+  5°  C.,  is  rapid  at  20°  C.  but  best  at  30°-38°  C. 

2.  Characters  of  growth  on  culture  media,     (a)  Broth. — The  growth  of  the 
plague  bacillus  in  broth  is  similar  to  that  of  some  streptococci :  the  medium 
is  clear  while  minute  flakes  adhere  to  the  walls  and  subsequently  fall  to  the 
bottom  of  the  tube.     Occasionally  a  pellicle  is  formed  on  the  surface.     Some- 
times— and  especially  when  the  broth  is  sown  from  a  previous  broth  culture— 
a  more  or  less  marked  turbidity  occurs. 

The  best  medium,  according  to  Yersin,  consists  of  an  alkaline  solution  of  peptone 
(2  per  cent.)  containing  1  to  2  per  cent,  of  gelatin. 

In  broth  culture  the  bacillus  will,  under  suitable  conditions,  give  rise  to 
stalactites  (p.  473). 

[To  obtain  good  stalactites  in  a  plague  culture  the  Advisory  Committee 
point  out  that  the  first  essential  is  an  absolute  lack  of  vibration  of  the  shelf 
on  which  the  flask  stands.  The  addition  of  oil  is  an  advantage  but  not  an 
essential.  A  neutral  broth  was  used  and  in  a  typical  stalactite  growth  the 

2o 


466  THE  PLAGUE   BACILLUS 

broth  remained  clear.  "  A  highly  characteristic  appearance  is  obtained 
when  1  c.c.  of  blood  containing  say  10  to  100  bacilli  per  c.c.  is  inoculated  into 
a  100  c.c.  flask  of  neutral  broth.  The  plasma  forms  a  soft  clot  dispersed 
throughout  the  broth  and  if  the  flask  be  kept  undisturbed  each  bacillus 
ultimately  gives  rise  to  a  tack-like  growth  enclosed  in  a  similarly  shaped 

cavity."     In  some  cases,  with  a  virulent  culture  the  broth 

was  somewhat  turbid.  ] 

(b)  Gelatin. — The  plague  bacillus  does  not  liquefy  gelatin. 
Isolated  colonies  appear  in  2-4  days  :  they  are  rounded, 
granular,  and  yellowish  and  are  occasionally  surrounded 
by  a  transparent   ring  with  irregular  margins.     In  stab 
culture   a  yellowish,  semi-transparent   growth   forms   on 
the  surface  while  a  whitish  streak  marks  the  line  of  the 
stab. 

(c)  Agar.  Glycerin-agar.  Serum. — When  a  scraping  from 
a  bubo  is  sown  on  any  of  these  media  the  plague  bacillus 
grows  in  the  form  of  transparent  white  colonies,  iridescent 
at  the  margins  when  examined  by  reflected  light.     When 
sub-cultured,  a  milky-white  slimy  layer  forms  in  24  hours. 

[The  growth  of  the  plague  bacillus  on  agar  is  very  like  that  of 
the  pasteurella  group  (Chap  XXVIII.)  and  it  is  only  by  further 
cultivation  and  inoculation  experiments  that  it  can  with 
certainty  be  distinguished  from  the  latter  bacillus.] 

(d)  Milk. — The  growth  is  poor  and  the  medium  is  not 
coagulated. 

(e)  Potato. — The  growth  is  slow  and  minimal  in  amount, 
consisting  of  a  whitish  or  yellowish  streak. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Viability  and  virulence. 

(2adS?).  fr°m  a  bUb°  In  cult'ure>  the  plague  bacillus  is  a  delicate  organism. 
A  temperature  of  58°  C.  for  an  hour  or  of  100°  C.  for  a 
minute  is  sufficient  to  sterilize  the  growth  ;  similarly,  exposure  to  sunlight 
for  3-4  hours  and  weak  antiseptic  solutions  are  equally  bactericidal. 

In  dried  pus,  the  organism  is  more  resistant  and  may  retain  both  its  vitality 
and  virulence  for  several  weeks. 

In  soil  the  bacillus  remains  alive  for  several  months  ;  its  virulence  under 
such  conditions  becomes  lowered,  but  can  be  restored.  According  to  Yokote, 
putrefaction  destroys  the  plague  bacillus  in  dead  bodies  in  15-30  days 
(p.  463). 

[The  Advisory  Committee  showed  that  floors  of  cow-dung  grossly  con- 
taminated with  B.  pestis  remain  infective  for  48  hours  while  floors  of  chunam 
(a  mixture  of  sand  and  lime  put  down  moist  and  allowed  to  set)  do  not 
remain  infective  even  for  24  hours  (the  infectivity  in  both  cases  was  tested 
by  rubbing  scrapings  into  susceptible  animals).] 

The  virulence  of  the  organism  diminishes  rapidly  in  artificial  culture. 
Scrapings  of  buboes  sown  on  agar  yield  colonies  varying  in  virulence  (Yersin) : 
the  larger  are  only  slightly  virulent  and  grow  so  much  more  rapidly  than  the 
virulent  colonies  that  the  latter  soon  become  crowded  out  with  the  result  that 
subsequent  sub-cultures  rapidly  lose  their  virulence.  (It  has  already  been 
pointed  out  that  it  is  possible  to  raise  the  virulence  of  an  attenuated  bacillus.) 

[The  virulence  of  Bacillus  pestis  for  the  rat  is  unaltered  (neither  increased 
nor  diminished)  when  passed  through  a  large  number  of  animals  in  succession. 


BIOLOGICAL  PROPERTIES  467 

The  Advisory  Committee  found  that  twenty-six  passages  from  rat  to  rat 
(M.  rattus  and  M.  decumanus),  by  sub-cutaneous  inoculation  without  inter- 
mediate culture,  occupying  in  all  89  days,  had  no  effect  on  the  virulence 
of  the  organism.  Similarly  its  virulence  was  unaltered  by  passage  through 
rats  by  cutaneous  inoculation  without  intermediate  culture.  These  experi- 
ments, however,  demonstrated  a  varying  susceptibility  of  the  Bombay  rat 
to  plague.] 

2.  Bio-chemical  reactions. 

[Media  containing  carbohydrates  :  The  plague  bacillus  produces  acid  but 
no  gas  in  glucose,  Isevulose,  galactose,  maltose,  mannite  and  dextrin  while 
in  lactose,  saccharose,  raffinose,  sorbite,  adonit,  inulin,  and  dulcite  no  acid 
is  formed  (MacConkey  and  others). 

[  The  plague  bacillus  produces  no  change  of  colour  in  litmus  milk. 

[In  media  containing  sodium  taurocholate  the  plague  bacillus  grows 
well.] 

3.  Toxin. 

Filtered  cultures  of  the  plague  bacillus  are  only  slightly  toxic  (Yersin  ; 
Calmette  and  Borrel).  Markl  showed  that  the  toxin  was  adherent  to  the 
bodies  of  the  bacilli,  so  that  in  order  to  obtain  it  cultures  several  weeks  old 
[in  which  the  toxin  had  diffused  into  the  medium]  had  to  be  used. 

Roux  prepared  a  very  toxic  product  from  a  bacillus  which  had  been 
increased  in  virulence  by  growing  it  in  collodion  sacs  in  the  peritoneal  cavities 
of  guinea-pigs.  This  bacillus  when  grown  in  0*5  per  cent,  gelatin-broth 
yielded  a  toxin  which  killed  mice  in  less  than  12  hours  when  inoculated  in 
quantities  of  0'014  c.c.,  but  which  had  very  little  effect  on  rabbits  and 
guinea-pigs.  Cultures  grown  in  this  way  were  macerated  under  toluol  for 
several  weeks  then  filtered  through  paper  and  precipitated  with  ammonium 
sulphate  ;  by  these  means  a  powder  was  obtained  of  which  the  fatal  dose  for 
a  mouse  was  O25  mg. 

Plague  toxin  is  a  very  unstable  substance.  Its  toxicity  is  diminished  by 
exposure  to  a  temperature  of  70°  C.  and  is  rapidly  destroyed  by  light  and 
air. 

Besredka  isolated  a  plague  endotoxin  by  using  a  method  similar  to  that  adopted 
by  him  for  the  preparation  of  typhoid  endotoxin.  The  bacilli  are  dried  and  triturated 
with  salt,  water  is  then  added  and  the  mixture  allowed  to  macerate  for  12  hours. 
After  centrifuging,  the  supernatant  liquid  contains  the  endotoxin.  The  latter  is 
thermolabile  and  is  fatal  to  mice  in  doses  of  0*006  c.c. 

[Rowland's  toxin. — Washing  living  plague  bacilli  with  chloroform  water 
while  killing  the  cell  removes  a  certain  amount  of  a  nucleo-protein  (substance 
A)  but  only  traces  of  the  substances  which  are  toxic  and  immunizing  for  rats. 
Organisms  which  have  been  thus  treated  are  toxic  and  possess  immunizing 
power  for  rats.  By  appropriate  treatment  (vide  infra)  a  further  nucleo- 
protein  (substance  B)  can  be  dissolved  out  which  is  more  toxic  and  more 
highly  immunizing  for  rats  than  substance  A  and  moreover  bacilli  from 
which  this  substance  has  been  removed  are  no  longer  toxic  or  immunizing 
for  these  animals. 

[Preparation. — The  bacillus  is  grown  on  a  neutral  lemco-peptone-agar  in  Roux 
flasks,  incubated  at  32°  C.  for  4  days  and  after  being  sterilized  with  chloroform 
vapour  the  growth  is  emulsified  in  10  c.c.  dilute  saline  solution.  The  emulsion  is 
centrifuged  and  the  deposit  re-emulsified  in  salt  solution,  filtered  through  fine  linen 
and  again  centrifuged.  This  second  deposit  is  pounded  in  a  mortar  with  anhydrous 
sulphate  of  soda  until  a  dry  powder  is  obtained.  The  powder  is  left  in  the  ice-chest 
overnight,  then  warmed  to  37°  C.,  well  stirred  and  replaced  in  the  ice-chest.  The 
freezing  and  thawing  is  repeated  several  times.  Water  is  then  added  to  make 


468  THE   PLAGUE   BACILLUS 

a  saturated  solution  at  37°  C.     The  bacterial  bodies  are  filtered  off  through  hardened 
paper  at  37°  C   and  suspended  in  water.     This  suspension  constitutes  solution  B. 

[The  lethal  dose  for  rats  is  an  amount  of  solution  corresponding  to  about  0*1  mg. 
of  contained  nucleo-protein.  Heating  to  55°  C.  lowers  its  toxicity.] 

4.  Vaccination. 
A.  Animals. 

(i)  With  toxins. — (a)  The  inoculation  of  even  highly  active  toxins  does  not 
result  in  the  production  of  an  absolute  and  lasting  immunity  and  does  not 
lead  to  the  formation  of  an  anti-plague  serum  (Roux  ;  Yersin  ;  Calmette 
and  Borrel). 

[(&)  A  single  injection  of  even  minute  quantities  (O'02-O'OOOl  mg.)  of 
Rowland's  toxin  confers  a  substantial  immunity  upon  rats.] 

(ii)  With  dead  cultures. — Inoculation  with  bacilli  killed  by  heat  gives 
better  results.  Yersin,  and  Calmette  and  Borrel  scraped  the  growth  from 
a  48-hour  old  culture  on  agar,  mixed  it  with  a  little  broth,  put  the  mixture 
into  tubes,  sealed  them  and  heated  them  to  58°  C.  for  1  hour,  and  in  this 
way  obtained  a  product  which  killed  rabbits  when  inoculated  in  large  doses 
into  the  veins  or  into  the  peritoneum.  If,  however,  one  or  two  inoculations 
of  sub -lethal  doses  were  given  into  the  veins  or  peritoneal  cavity  the  animal 
was  protected  against  the  subsequent  sub-cutaneous  inoculation  of  a  living 
virulent  bacillus,  provided  that  at  the  time  of  the  test  inoculation  the  animal 
had  completely  recovered  from  the  effects  of  the  vaccine. 

Rabbits  can  also  be  immunized  by  sub-cutaneous  inoculation  of  heated  cultures 
but  the  method  requires  time  :  as  a  rule  it  is  necessary  to  give  three  or  four  inocula- 
tions at  intervals  of  a  fortnight. 

The  guinea-pig  is  not  so  readily  immunized  by  this  method. 

(iii)  With  living  cultures. — Horses  are  difficult  to  immunize.  The  inocula- 
tion of  bacilli  killed  by  heat  produces  little  reaction  and  is  very  slow  in  its 
results  :  the  most  efficient  method  is  to  inoculate  into  the  veins  first  heated 
cultures,  then  progressively  increasing  doses  of  living  bacilli  (Roux).  The 
method  of  immunizing  horses  for  the  supply  of  therapeutic  serum  actually 
in  use  at  the  Pasteur  Institute,  Paris,  is  described  later  (vide  Serum  therapy). 

B.  Man. 

(i)  With  dead  cultures. — Haffkine  was  the  first  to  prepare  a  vaccine  for 
human  prophylaxis  by  killing  virulent  cultures  by  heat  at  60°  C. 

The  bacillus  was  sown  in  large  flasks  half-filled  with  a  special  broth  (a  maceration 
of  goat  meat  peptonized  with  hydrochloric  acid  and  neutralized)  or  ordinary  broth,, 
the  surface  of 'the  latter  being  covered  with  a  thin  layer  of  sterilized  oil  to  obtain 
a  growth  of  stalactites  (p.  473).  The  flasks  were  incubated  [in  large  room?]  at 
27°-30°  C.  [the  average  temperature  in  Bombay]  for  2  months,  being  shaken  from 
time  to  time  in  order  to  break  up  the  stalactites.  After  verifying  the  purity  of  the 
culture  the  contents  were  distributed  into  tubes  which  were  sealed  and  heated  at 
60°  C.  for  a  quarter  of  an  hour ;  a  little  carbolic  acid  (0'5  per  cent.)  was  subsequently 
added.  The  vaccine  was  used  2  months  after  preparation. 

For  purposes  of  human  vaccination  5  c.c.  of  the  vaccine  are  inoculated 
beneath  the  skin  of  the  arm  in  the  neighbourhood  of  the  insertion  of  the 
deltoid.  (Inoculation  in  the  region  of  the  shoulder  or  of  the  abdomen  is 
less  painful.)  A  few  hours  later  a  painful  swelling  occurs  around  the  site  of 
inoculation  accompanied  by  a  rise  of  temperature  (38°  C.)  and  a  slight  swelling 
of  the  glands  ;  the  temperature  is  again  normal  in  about  36  hours  and  the 
symptoms  have  disappeared.  Immunity  is  established  as  soon  as  the  tem- 
perature falls.  The  vaccine  exerts  no  prophylactic  properties  during  the 


VACCINATION  469 

period  of  reaction  following  inoculation  and  if  the  patient  is  in  the  incubation 
period  of  the  disease,  the  latter  runs  its  ordinary  course,  though  it  would 
appear  that  under  such  conditions  the  vaccine  may  have  some  favourable 
influence  on  it.  The  immunity  lasts  for  one  year  at  least  (Haffkine),  and  it 
would  appear  to  be  more  efficient  and  to  last  longer  in  Europeans  than  in 
Hindus.  Haffkine  has  inoculated  some  hundreds  of  thousands  of  individuals 
with  his  vaccine. 

Jatta  and  Maggiora  prepare  a  vaccine  similar  to  that  of  Haffkine.  Plague  bacilli 
are  grown  in  very  shallow  layers  of  broth  for  4  days :  the  culture  is  then  heated  to 
65°  C.  and  carbolic  acid  added.  The  dose  is  1  c.c. 

Gosio  grows  the  bacillus  in  a  shallow  layer  of  broth.  The  growth  is  precipitated 
with  a  powerfully  agglutinating  serum,  collected,  made  into  an  emulsion  and 
sterilized  by  heating  at  65°  C.  for  an  hour.  (To  ensure  the  sterility  of  the  vaccine 
a  little  is  sown  in  broth  containing  potassium  tellurate  (1  part  in  100,000) ;  if  the 
vaccine  be  sterile  the  appearance  of  the  broth  remains  unchanged,  if  on  the  other 
hand  blackish  flakes  appear  in  it  then  the  sterilization  is  imperfect.)  Each  c.c.  of 
culture  yields  about  1  mg.  of  vaccine  of  which  the  vaccinating  dose  for  an  adult 
man  is  2-3  mg.  (2-3  c.c.  of  emulsion).  In  this  vaccine  the  antiserum  merely  plays 
a  mechanical  part  in  precipitating  the  bacilli ;  its  antitoxic  properties  are  destroyed 
at  the  temperature  (65°  C.)  at  which  the  vaccine  is  sterilized. 

The  German  Plague  Commission  (Gaffky,  Pfeiffer,  Sticker  and  Dieudonne) 
proved  the  superior  efficacy  of  vaccines  prepared  from  cultures  on  solid 
media. 

The  bacillus  is  sown  on  agar  in  Roux  bottles,  and  incubated  for  3  days  and  the 
growth  scraped  off.  The  emulsion  is  then  heated  for  three-quarters  of  an  hour 
at  65°  C.,  and  sufficient  sterile  normal  saline  solution  added  to  make  the  total  volume 
up  to  200  c.c.  for  the  growth  from  each  bottle.  The  emulsion  is  distributed  into 
tubes,  sealed  and  heated  a  second  time  to  ensure  its  sterility.  One  c.c.  contains  about 
2 '5  mg.  of  bacilli. 

The  German  vaccine  has  been  used  for  the  inoculation  of  200,000  Japanese. 
A  similar  vaccine,  prepared  at  the  Manguinhos  Institute,  Rio  de  Janeiro,  has 
proved  of  considerable  value  in  Brazil. 

(ii)  With  living  attenuated  cultures. — In  some  old  agar  cultures  of  the 
plague  bacillus,  Yersin  and  Carre  found  a  number  of  colonies  of  an  attenuated 
bacillus  (Race  6)  which  only  proved  fatal  to  20  per  cent,  of  rats  inoculated 
and  in  the  other  cases  afforded  immunity.  Yersin  inoculated  himself  with 
this  attenuated  virus  and  merely  suffered  from  a  slight  febrile  attack. 

Kolle  and  Otto  proposed  to  use  as  a  vaccine  a  bacillus  of  low  virulence 
attenuated  by  growing  it  at  40°— 41°  C.  This  virus  is  not  fatal  to  guinea-pigs 
when  inoculated  in  quantities  of  two  loopsful  sub-cutaneously,  and  produces 
an  immunity  lasting  several  months  in  guinea-pigs  and  rats.  On  these  data, 
Strong  has  shown  that  any  plague  bacillus  which  does  not  kill  guinea-pigs 
(250  grams)  when  a  whole  agar  tube  culture  is  inoculated  sub-cutaneously 
can  be  used  as  a  vaccine,  and  he  has  inoculated  200  persons  in  the  Philippines 
each  with  a  tube  of  such  a  culture  without  producing  any  disturbance  of 
health. 

(iii)  With  heated  exudates. — Terni  and  Bandi  use  the  heated  peritoneal 
exudate  of  a  plague-infected  guinea-pig  as  a  vaccine.  This  method  of  vaccina- 
tion is  said  to  give  a  high  degree  of  immunity  in  a  short  space  of  time  and  to 
produce  a  merely  insignificant  reaction.  It  has  up  till  now  been  little  used 
in  practice  though  a  few  hundred  persons  were  inoculated  at  Rio  de  Janeiro. 

A  culture  of  a  virulent  plague  bacillus  is  inoculated  into  the  peritoneal  cavity  of 
a  guinea-pig,  and  when  the  animal  is  in  extremis  it  is  killed.  The  peritoneal  exudate 
is  collected,  and  diluted  with  a  little  normal  saline  solution ;  the  mixture  is  incubated 
for  12  hours  at  37°  C.  and  then  heated  at  50°  C.  for  2  hours  on  two  consecutive  days. 


470  THE  PLAGUE   BACILLUS 

The  exudate  from  one  guinea-pig  is  made  up  to  about  50  c.c.  with  the  following 
solution : 

Water,     -  -         100        c.c. 

Sodium  chloride,       -  0'70  gram. 

Sodium  carbonate,    -  0*25      „ 

Carbolic  acid  crystals,        -         -  0'50     „ 

The  dose  of  vaccine  for  a  man  is  2-2'5  c.c. 

(iv)  With  bacillary  extracts. — (a)  Lustig  and  Galeotti  have  introduced  the 
use  of  bacterial  extracts  as  a  method  of  immunization  against  plague.  The 
technique  is  complicated  and  the  results  do  not  show  that  the  method  has  any 
advantage  over  the  preceding  :  the  immunity  is  of  but  short  duration.  Some 
200  persons  were  inoculated  at  San  Nicola  de  la  Plata.  Tavel  of  Berne 
has  made  some  modifications  in  the  technique. 

In  Lustig  and  Galeotti's  method  the  bacilli  are  sown  on  agar  plates  and  incubated 
for  3  days ;  the  growth  is  then  scraped  off  and  allowed  to  macerate  in  a  1  per  cent, 
solution  of  caustic  potash  for  24  hours.  The  solution  is  diluted,  filtered  and  then 
precipitated  with  dilute  acetic  acid  and  the  precipitate  washed  and  finally  dried 
in  vacuo. 

The  precipitate  consisting  of  nucleo-proteins  is  used  for  human  vaccination  in 
doses  of  0'003  gram  dissolved  in  a  0'5  per  cent,  solution  of  sodium  carbonate.  The 
inoculation  is  painful  and  is  accompanied  by  a  sharp  reaction. 

(v)  Sero- vaccination. — With  the  various  methods  of  vaccination  described 
above  there  is  a  delay  of  a  few  days  before  the  person  inoculated  becomes 
immune,  and  in  some  cases  the  person  so  inoculated  is  more  susceptible  during 
that  time  to  the  plague  bacillus  :  in  other  words,  a  negative  phase  follows 
vaccination.  To  overcome  this  difficulty  Calmette  and  Salimbeni  devised 
a  method  of  sero-vaccination.  Either  a  mixture  of  vaccine  and  antiplague 
serum  is  inoculated,  or  a  dose  of  serum  (5  c.c.)  followed  2  days  later  by  a 
dose  (2-3  c.c.)  of  Haffkine's  vaccine.  In  this  way  not  only  is  the  person 
immunized  at  once  but  the  reaction  following  the  inoculation  is  less  marked. 
On  the  other  hand,  unfortunately,  the  immunity  is  only  short-lived  and 
the  results  "  are  not  much  better  than  those  obtained  by  the  use  of  serum 
alone  "  (Besredka). 

To  prepare  the  vaccine  the  bacillus  is  sown  on  agar  in  Roux  bottles  and  incubated 
for  48  hours.  The  growth  is  then  scraped  off,  mixed  with  normal  saline  solution, 
and  the  emulsion  filtered  through  filter  paper.  The  organisms  retained  on  the  filter 
are  made  into  an  emulsion  with  a  little  normal  saline  solution,  heated  at  70°  C.  for 
an  hour,  and  dried  in  vacuo.  The  product  mixed  with  antiplague  serum  constitutes 
the  vaccine. 

Shiga  prepares  a  vaccine  in  the  following  manner  : 

Agar  cultures  three  days  old  are  scraped  off,  made  into  an  emulsion  with  normal 
saline  solution  (1  c.c.  for  each  loopful  of  growth),  heated  at  60°  C.  for  half  an  hour 
and  carbolic  acid  added  in  the  proportion  of  0'5  per  cent.  Just  before  use, 
equal  parts  of  the  emulsion  and  antiplague  serum  are  mixed  and  inoculated  in  doses 
of  O'6-l  c.c.  for  a  man  :  a  few  days  later  an  inoculation  of  emulsion  without  serum 
is  administered.  This  sero-vaccine  has  been  used  in  Japan. 

Besredka's  vaccine.— Besredka  attributed  the  poor  results  obtained  by 
Calmette's  method  to  the  presence  of  an  excess  of  serum  in  the  mixture.  By 
reducing  the  amount  of  serum  to  a  minimum  this  observer  was  able  to  induce 
a  rapid,  powerful  and  lasting  immunity  in  animals  (p.  382). 

Bacilli  from  a  two-day-old  culture  on  agar  are  made  into  an  emulsion  with  a  very 
little  normal  saline  solution,  heated  at  60°  C.  for  an  hour  and  then  mixed  with  anti- 
plague  serum.  At  the  end  of  24  hours  the  bacilli  after  being  washed  repeatedly 
to  remove  all  traces  of  free  serum  are  made  into  an  emulsion  with  normal  saline 
solution,  distributed  into  tubes,  sealed  and  then  heated  again  for  an  hour  at  54°  C. 
to  ensure  sterility.  The  vaccine  is  now  ready  for  use. 


SERUM  THERAPY  471 

The  vaccine  has  no  toxic  properties  and  gives  rise  to  no  symptoms  in  mice  and 
guinea-pigs.  Inoculated  mice  are  immune  in  48  hours  and  the  immunity  has  been 
shown  to  last  for  five  months  and  a  hah0. 

For  man  the  dose  is  the  amount  of  emulsion  corresponding  to  5  mg. 
of  dead  bacilli.  The  vaccine  has  been  used  in  clinical  practice  in  Peru  and 
Mexico. 

5.  Serum  therapy. 

The  serum  of  persons  who  have  recovered  from  plague  has  slight  prophy- 
lactic and  curative  properties  (Metin). 

Similarly,  the  serum  of  rabbits  immunized  against  the  bacillus  also  shows 
therapeutic  and  prophylactic  properties.  A  rabbit,  for  instance,  can  be  pro- 
tected against  an  experimental  infection  with  a  virulent  virus  by  the  inocula- 
tion of  3  c.c.  of  such  serum  sub-cutaneously,  and  in  a  rabbit  which  has  already 
been  infected  the  disease  can  be  arrested  and  the  animal  cured  by  the  admini- 
stration of  the  same  dose  of  serum — provided  that  the  latter  be  treated 
within  12  hours  of  the  infecting  inoculation. 

Yersin's  serum. — The  serum  of  horses  immunized  by  Roux's  method  (vide 
ante)  and  collected  3  weeks  after  the  last  inoculation  exhibits  immunizing, 
therapeutic  and  antitoxic  properties. 

(a)  Preparation  of  the  serum.  Method  adopted  at  the  Pasteur  Institute  in 
Paris. — The  bacillus  used  is  a  fully  virulent  bacillus  of  human  origin  kept  virulent 
by  frequent  passage  through  guinea-pigs  and  rats.  The  bacillus  is  sown  on  agar 
in  Roux  bottles  and  incubated  for  3  days,  the  growth  is  then  scraped  off,  made  into 
an  emulsion  with  normal  saline  solution  and  filtered  through  absorbent  wool.  The 
homogeneous  filtrate  is  heated  at  65°  C.  for  1  hour. 

The  first  inoculation  consisting  of  a  small  quantity  of  heated  emulsion  (about 
T/^th  of  a  bottle  culture  or  |th  of  a  tube  culture)  is  given  into  the  jugular  vein  of 
the  horse.  The  horse  reacts  sharply  and  has  a  marked  rise  of  temperature  during 
the  next  48  hours.  Sometimes  the  inoculation  is  followed  immediately  by  severe 
syncopal  attacks  from  which  the  animal  may  die. 

A  second  inoculation  is  given  a  fortnight  later  and  after  that  the  inoculations  are 
repeated  at  intervals  of  a  week,  the  amount  of  material  inoculated  being  pro- 
gressively increased  until  a  whole  bottle  culture  is  administered  at  one  inoculation. 
When  the  serum  exhibits  immunizing  properties  heated  cultures  are  superseded  by 
living  hyper- virulent  bacilli,  the  initial  dose  being  about  ^jth  of  a  bottle  culture. 
The  animals  lose  a  good  deal  of  weight  during  immunization  and  the  inoculations 
must  not  be  pressed  unduly.  Immunization  occupies  some  6-8  months  and  the 
horse  is  not  bled  until  10  days  or  a  fortnight  after  the  last  inoculation.  The  immunity 
is  maintained  by  administering  in  the  intervals  between  bleeding  two  inoculations 
of  one- half  and  a  whole  bottle  of  culture  respectively  at  an  interval  of  a  week. 
Before  leaving  the  Institute  the  serum  is  heated  on  three  separate  occasions  at  54°  C. 
to  diminish  its  toxic  properties. 

(6)  Properties  of  the  serum. — Yersin's  antiplague  serum  exhibits  immunizing  and 
therapeutic  properties.  If  injected  previously  to  an  experimental  inoculation  the 
development  of  the  disease  in  susceptible  animals  is  prevented.  If  given  after  an 
infecting  inoculation  the  course  of  the  disease  is  interrupted  :  the  longer  the  time 
which  is  allowed  to  elapse  between  infection  and  the  administration  of  serum,  the 
larger  must  be  the  dose  of  serum  and  the  smaller  are  the  chances  of  recovery.  In 
guinea-pigs  infected  through  a  shaved  area  of  skin  the  serum  has  no  therapeutic 
properties  even  though  administered  one  hour  after  the  infection.  The  Pasteur 
Institute  serum  cures  mice  infected  by  the  bite  of  an  insect  if  inoculated  in  doses 
of  O'l  c.c.  sixteen  hours  after  infection.  The  immunity  following  the  inoculation  of 
serum  lasts  but  a  very  short  time,  some  ten  days  or  so. 

(c)  Human  serum  therapy. — The  efficacy  of  antiplague  serum  in  the  treatment 
of  plague  in  the  human  subject  is  shown  by  the  experience  of  Yersin,  Calmette  and 
Salimbeni,  Metin  and  others. 

(i)  Prophylaxis. — The  inoculation  of  10  c.c.  of  the  serum  prepared  at  the  Pasteur 
Institute  affords  immunity  to  the  disease  at  once,  but  the  immunity  only  lasts  about 


472  THE  PLAGUE   BACILLUS 

ten  days  which  is  a  great  disadvantage  in  practice,  so  that  in  the  majority  of  cases  it  is 
necessary  to  resort  to  vaccines  (vide  ante}. 

(ii)  Curative. — In  treating  cases  of  plague  the  inoculation  of  serum  should  be 
repeated  at  intervals,  the  total  volume  to  be  administered  being  from  200  to 
400  c.c.  It  may  be  inoculated  beneath  the  skin,  but  intra- venous  inoculation  gives 
far  better  results.  As  with  all  other  serums,  the  sooner  it  is  used  the  better  the 
chance  the  patient  has  of  recovering.  The  longer  the  administration  is  delayed  the 
larger  must  be  the  initial  dose  of  serum  given.  Generally  speaking  it  is  sufficient  to 
inoculate  40-60  c.c.  as  soon  as  possible  into  a  vein  and  to  give  two  further  doses 
(sub-cutaneously  of  20-^40  c.c.  each  time)  within  the  next  24  hours.  Daily  inocula- 
tions of  10-40  c.c.  should  also  be  given  sub-cutaneously  until  the  temperature  has 
fallen  to  normal.  In  severe  cases  Penna  obtained  good  results  by  giving  at  the 
outset  100  c.c.  in  the  veins  followed  by  a  daily  inoculation  into  the  veins  of 
60—100  c.c.  This  method  is  to  be  strongly  recommended,  many  of  the  failures 
recorded  being  due  simply  to  the  fact  that  the  serum  has  been  used  in  too  small  doses 
and  has  been  given  exclusively  beneath  the  skin.  It  is  important  also  not  to  stop 
the  administration  of  serum  suddenly  when  the  fever  has  subsided  but  to  continue 
its  use  for  several  days  in  gradually  diminishing  doses. 

[(&)  Rowland  prepares  a  serum  which  when  tested  on  rats  exhibits  immuniz- 
ing, antitoxic  and  curative  properties. 

[A  horse  is  inoculated  on  several  successive  occasions  with  Rowland's  "  solution 
B  "  prepared  as  above  (vide  Toxins).  The  immunizing  process  lasted  over  a  period 
of  6  months.  The  initial  dose  administered  was  0*01  mg.  and  the  final  dose  240  mg. 
The  horse  was  bled  twenty- one  days  after  the  last  inoculation.  The  local  reaction 
following  inoculation  was  very  similar  to  that  following  the  inoculation  of  diphtheria 
toxin  :  a  varying  amount  of  swelling  and  oedema  with  transitory  constitutional 
disturbance  and  a  little  temperature  reaction.  There  was  no  tendency  to  abscess 
formation  or  to  the  huge  hard  swellings  which  not  infrequently  supervene  upon 
the  inoculation  of  unfiltered  cultures. 

[The  serum  neutralizes  the  toxin  in  the  immunizing  solution  ;  1'25  c.c.  of 
serum  neutralize  100  lethal  doses  of  toxin  for  the  rat.  A  serum  prepared  by 
Yersin's  method  has  no  antitoxic  action  on  the  toxin. 

[In  doses  of  O'l  c.c.  the  serum  protects  rats  against  a  subsequent  inoculation 
of  the  standard  test  dose  of  virulent  culture  given  the  following  day. 

[Administered  in  doses  of  0'5  c.c.  sub-cutaneously  six  hours  after  inoculation 
of  a  living  plague  culture  and  on  the  opposite  side  of  the  body  to  the  latter  the 
death  rate  among  rats  was  reduced  from  80  per  cent,  to  18  per  cent,  and  in 
those  cases  in  which  the  treated  rats  died  the  length  of  life  was  prolonged 
from  three  to  five  days  to  ten  days.  In'  a  comparative  experiment  with 
Yersin's  serum  two  rats  only  out  of  ten  survived  while  of  the  ten  treated  with 
Rowland's  serum  all  survived  and  the  ten  controls  which  received  no  serum 
at  all  all  died.] 

6.  Agglutination. 

Plague  serum  (in  dilutions  of  1  in  50  to  1  in  500)  agglutinates  broth  cultures 
of  the  plague  bacillus.  The  degree  of  agglutinability  of  the  plague  bacillus 
depends  upon  the  consistency  of  the  culture  and  not  on  its  virulence 
(Shibayama). 

The  agglutination  of  the  bacillus  by  the  blood  of  persons  suffering  from 
plague  is  feeble  and  inconstant.  In  most  cases  it  can  only  be  effected  with 
dilutions  of  1  in  5  or  1  in  20  ;  rarely  it  may  be  observed  in  a  dilution  of  1  in  40. 
The  agglutinating  property,  which  hardly  ever  appears  before  the  end  of  the 
first  week  of  the  disease,  is  most  marked  in  the  blood  of  convalescents  and 
cannot  therefore  be  of  any  great  help  in  the  diagnosis  of  plague  (Zabolotny 
and  Cairus)  though  it  may  be  useful  in  diagnosing  cases  unrecognized  in  the 
early  stages  of  the  disease. 


ISOLATION   OF  THE  BACILLUS  473 

7.  Precipitins. 

[The  addition  of  plague  serum  to  a  filtrate  of  the  plague  bacillus  produces 
first  a  cloudiness  then  an  abundant  precipitate  which  settles  to  the  bottom  of 
the  tube  leaving  the  supernatant  liquid  clear.] 

[A  filtrate  of  the  B.  pseudo-tuberculosis  rodentium  added  to  plague  serum  gives  a 
similar  but  less  abundant  precipitate  and  the  fluid  takes  much  longer  to  clear.  ] 

SECTION  IV.— THE  ISOLATION  AND  IDENTIFICATION  OF  THE 
BACILLUS.— POST  MORTEM  APPEARANCES  IN  NATURALLY 
INFECTED  RATS. 

In  the  living  subject  the  pus  of  the  buboes,1  the  juice  of  the  lymphatic 
glands,  the  blood  (obtained  by  pricking  the  finger  or  lobe  of  the  ear),  sputum, 
[urine,]  and  the  fluid  in  the  petechise  must  be  examined  for  the  presence 
of  the  bacillus. 

Even  when  there  is  an  absence  of  buboes,  the  bacillus  is  present  in  the 
lymphatic  glands  :  in  such  cases,  remove  a  gland  and  examine  it  as  detailed 
below. 

In  the  dead  body  the  spleen,  lungs,  kidneys,  etc.  should  be  examined. 

The  technique  of  identification  of  the  bacillus  is  as  follows  : 

1.  Microscopical  examination. — Prepare  films  on  slides,  fix  in  alcohol-ether, 
stain   with   carbol-thionin   or   carbol-violet.     Stain   other  films   by   Gram's 
method— the  plague  bacillus  is  gram-negative. 

[Microscopical  examination  alone  cannot  be  relied  upon  for  the  recognition 
of  the  plague  bacillus  :  the  B.  pseudo-tuberculosis  rodentium,  organisms  of 
the  pasteurella  and  often  of  the  salmonella  group  are  indistinguishable  from 
the  plague  bacillus  under  the  microscope.] 

2.  Cultures. — Sow  the  gland  pulp  or  scrapings  from  the  viscera  on  agar 
and  incubate  at  37°  C. 

[To  examine  the  blood  collect  2  c.c.  of  blood  by  means  of  a  sterile  syringe  from  a 
suitable  vein  at  the  bend  of  the  elbow  (p.  193)  and  distribute  in  small  quantities  on 
a  series  of  agar  slopes.  ] 

Haffkine  has  described  an  ingenious  and  rapid  method  for  the  identification  of 
cultures  of  the  plague  bacillus.  The  method  consists  in  sowing  the  suspected 
material  in  broth  on  the  surface  of  which  a  layer  of  sterilized  butter  or  oil  has  been 
poured  (p.  468).  Under  these  conditions  the  plague  bacillus  gives  origin  to  stalac- 
titic  forms  of  growth  suspended  from  the  lower  surface  of  the  oil.  [This  appearance 
is  seen  with  only  a  few  other  organisms,  namely :  the  bacilli  of  the  hsemorrhagic  sep- 
ticaemia group,  but  these  happen  to  be  just  the  organisms  which  are  likely  to  be 
confounded  with  the  plague  bacillus.  The  formation  of  stalactites  in  broth  culture 
cannot  therefore  alone  be  accepted  as  a  sufficient  diagnostic  feature.] 

3.  Inoculation  experiments. — Inoculate  a  loopful  of  growth  from  an  agar 
culture  beneath  the  skin  of  a  mouse  or  guinea-pig  or  into  the  nasal  fossse  of  a 
guinea-pig.     If  the  culture  be  a  growth  of  the  plague  bacillus  the  animal  will 
die  in  2-5  days  and  the  organism  can  be  recovered  from  the  blood,  spleen,  etc. 

When  dealing  with  material  containing  many  adventitious  organisms,  such 
as  stools,  decomposing  carcases  etc.,  it  is  best  to  rub  a  little  of  the  suspected 
material  into  a  previously  shaved  area  of  skin  of  a  guinea-pig  (p.  463). 

[In  examining  urine  for  the  presence  of  the  plague  bacillus,  the  Advisory 
Committee  adopted  both  the  cutaneous  and  the  sub-cutaneous  inoculation 
methods,  and  obtained  better  results  than  previous  observers  who  had  relied 
mainly  on  cultivation  methods.] 

1  In  bubonic  pus  the  plague  bacillus  is  occasionally  associated  with  staphylococci, 
the  colon  bacillus,  etc.  In  suppurating  buboes  the  specific  bacillus  may  have  disappeared. 


474  THE   PLAGUE   BACILLUS 

[4.  Differential  diagnosis. — The  differential  diagnosis  of  the  plague  bacillus 
from  the  organisms  most  likely  to  be  mistaken  for  it,  viz.  :  B.  pseudo-tubercu- 
losis rodentium,  the  bacilli  of  the  hsemorrhagic  septicaemia  group,  and  the 
bacilli  of  the  salmonella  group,  will  depend  upon  the  following  observations. 

[1.  The  inoculation  of  a  white  rat  will  exclude  the  B.  pseudo-tuberculosis 
rodentium  which  is  non -pathogenic  to  white  rats  but  gives  identical  ferment 
ation  reactions. 

[2.  The  characteristics  of  the  growth  in  media  containing  taurocholate  of 
sodium  will  differentiate  the  haemorrhagic  septicaemia  group  :  the  plague 
bacillus  grows  well  on  such  media  while  the  growth  of  the  latter  group  of 
organisms  is  inhibited. 

[3.  The  characteristics  of  the  growth  on  agar  and  in  broth  (absence  of 
stalactites)  and  the  fermentation  reactions  will  distinguish  the  plague  bacillus 
from  the  bacilli  of  the  salmonella  group.  ] 

Post-mortem  appearances  in  rats  naturally  infected  with  Plague.1 

The  diagnosis  of  spontaneous  plague  in  the  rat  is  a  matter  of  much  interest  [and 
the  following  is  a  brief  account  of  the  appearances  seen  post  mortem] : 

[Sub-cutaneous  congestion  is  not  infrequently  a  marked  feature :  it  may  be 
general  but  in  some  cases  is  limited  to  the  neighbourhood  of  the  bubo.  Sub -cutaneous 
haemorrhages  occur  in  about  40  per  cent,  of  rats  and  are  most  frequently  to  be  seen 
in  the  sub-maxillary  region.  Buboes  are  present  in  the  majority  of  cases  but  may 
be  absent  (15  per  cent.) ;  when  present  they  occur  in  the  majority  of  cases  in  a 
single  situation  and  most  commonly  in  the  neck.  The  liver  may  show  necrotic 
changes  which  have  the  appearance  of  an  excessive  deposit  of  fat,  and  a  condition 
of  the  greatest  importance  in  diagnosis  is  the  occurrence  of  small  necrotic  foci 
scattered  over  its  surface  and  throughout  its  substance.  The  spleen  is  firm  and 
does  not  collapse  like  a  soft  normal  spleen  ;  granules  or  nodules  may  be  well-marked 
in  it  and  may  be  confluent.  The  kidneys  and  supra-renal  capsules  are  often  con- 
gested. Haemorrhages  are  fairly  common  in  the  lungs  and  visceral  pleurae.  The 
presence  of  pleural  effusion  is  very  characteristic  and  of  great  value  in  diagnosis. 

[In  naturally  infected  plague  rats  the  most  important  features  for  purposes  of 
diagnosis  are : 

1.  A  typical  bubo — most  commonly  in  the  neck. 

2.  Granular  liver — not  seen  except  in  plague  rats. 

3.  Haemorrhages  beneath  the  skin  and  in  the  internal  organs  are  very  suggestive. 

4.  Pleural  effusion. 

In  putrid  rats,  bubo,  granular  liver  and  pleural  effusion  may  persist  and  are  of 
great  significance.] 

A  microscopical  examination  of  scrapings  of  buboes  and  spleen  and  inoculation 
tests  will  clinch  the  diagnosis. 

t1  This  account  is  abstracted  from  the  Reports  of  the  Advisory  Committee.  Journal  of 
Hygiene,  vii.  p.  324  et  seq.] 


CHAPTER  XXX. 

MICROCOCCUS  MELITENSIS.1 

Introduction. 

Section  I. — Experimental  inoculation,  p.  476. 

Man,  p.  476.     Animals,  p  476. 
Section  II. — Morphology,  p.  476. 

1.  Microscopical  appearance  and  staining  reactions,  p.  476.     2.  Cultural  charac- 
teristics, p.  476. 
Section  III. — Biological  properties,  p.  477. 

1.  Vitality,  p.  477.     2.  Biochemical  reactions,  p.  477.     3.  Toxins,  p.  477.     4.  Im- 
munity, p.  477.     Vaccination,  p.  477.     5.  Agglutination,  p.  478.     6.  Immune  body, 
p.  478. 
Section  IV. — Detection,  isolation  and  identification  of  the  organism,  p   478. 

BRUCE  gave  the  name  Mediterranean  fever  to  a  disease  which  is  very  common 
in  Malta,  and  which  had  been  mistaken  for  enteric  fever  or  malaria  until  he 
showed  that  it  is  a  specific  disease  due  to  a  specific  micro-organism,  the 
Micrococcus  melitensis. 

Mediterranean  fever  (Malta  fever,  undulant  fever)  occurs  along  the  whole 
of  the  Mediterranean  littoral,  in  India,  China,  England,  France,  and  other 
countries.  [Sir  David  Bruce  has  recently  recorded  an  interesting  and  localized 
epidemic  in  Central  Africa  to  which  the  inhabitants  had  given  the  name 
Muhinyo.  ] 

In  patients  who  have  died  of  the  disease  the  organism  is  found  in  pure  culture 
in  the  liver,  spleen  and  kidneys.  During  life  it  can  easily  be  obtained  by  puncturing 
the  spleen  of  infected  persons,  and  it  is  generally  present  in  the  urine  in  the  acute 
stage  of  the  disease  and  during  convalescence  (Durham).  It  only  occurs  in  the 
blood  in  small  numbers  and  then  mainly  during  the  febrile  attack. 

In  the  great  majority  of  cases  infection  takes  place  through  drinking 
infected  goats'  milk  (Bruce).  In  Malta,  goats  are  frequently  infected  with 
the  micrococcus  and  eliminate  the  organism  in  their  milk  ;  according  to 
Horrocks  and  Kennedy  this  is  normally  the  case  with  10  per  cent,  of  the 
Maltese  goats.  Direct  contact  with  the  sick  is  also  a  source  of  infection  and 
those  who  nurse  them  frequently  become  infected  (Manson)  :  handling 
infected  milk  and  urine  is  particularly  dangerous  especially  if  there  be  an 

f1  Though  generally  described  as  a  coccus  it  has  been  decided  to  place  this  organism 
among  the  gram-negative  bacilli  on  account  of  the  many  affinities  which  it  has  with  the 
gram-negative  bacilli  of  the  typhoid-colon  group  and  the  absence  of  affinities  with  the 
other  gram-negative  cocci.  ] 

[For  further  information  the  reader  is  referred  to  the  Reports  of  the  Commission 
for  the  investigation  of  Mediterranean  fever  (Harrison  &  Sons)  and  to  Eyre's  Milroy 
Lectures,  1908.] 


476  THE   COCCUS   OF  MEDITERRANEAN   FEVER 

abrasion  on  the  skin  (Shaw).  It  is  possible  that  infection  may  also  take 
place  through  dust  contaminated  with  the  coccus  settling  on  the  nasal  or 
ocular  mucous  membrane  (Shaw)  (vide  infra  experimental  inoculation). 
Zammitt  has  suggested  that  mosquitoes  may  act  as  carriers  of  the  infection. 

SECTION  I.—  EXPERIMENTAL  INOCULATION. 

Man.  —  Intentional  or  accidental  infection  of  men  with  cultures  of  the 
Micrococcus  melitensis  has  several  times  been  followed  after  an  incubation 
period  of  five  days  to  a  fortnight  by  a  typical  attack  of  Mediterranean  fever. 

Animals.  —  Monkeys  and  goats  are  highly  susceptible  to  the  disease. 

After  sub-cutaneous  inoculation  of  a  small  quantity  of  an  agar  culture 
rubbed  up  in  a  few  drops  of  sterile  water  monkeys  suffer  from  a  disease 
very  similar  to  that  in  man. 

At  the  close  of  an  incubation  period  of  2—5  days  the  temperature  rises  2°  or  3°  C. 
and  is  frequently  of  a  daily  remittent  character  ;  a  period  of  apyrexia  lasting  a  few 
days  followed  by  a  second  rise  of  temperature  often  intervenes  during  the  course  of 
the  disease.  The  serum  agglutinates  the  coccus  after  about  the  fifth  day  in  dilutions 
of  1  in  100  to  1  in  1000.  The  disease  may  last  several  months  and  ultimately  end 
in  recovery,  but  as  a  rule  the  animal  dies  about  the  end  of  the  second  week.  Post 
mortem,  the  liver  and  spleen  are  swollen  and  yield  pure  cultures  of  the  micrococcus. 
There  are  never  any  lesions  in  the  Peyer's  patches. 

By  means  of  feeding  experiments  Horrocks  and  Kennedy  infected  monkeys 
and  goats,  and  Shaw  produced  the  disease  in  monkeys  by  smearing  the  nasal 
and  ocular  mucous  membranes  with  cultures  and  infected  dust. 

Dogs,  horses,  asses  and  mules  are  also  susceptible  to  infection  with  the 
micrococcus. 

Rabbits,  guinea-pigs,  rats  and  mice  are  more  immune  than  the  preceding. 
Durham  and  Eyre  produced  a  fatal  result  in  these  animals  by  inoculating  them 
intra-cerebrally,  and  the  virulence  of  the  organism  is  found  to  be  rapidly 
increased  by  intra-cerebral  passage  through  rabbits  or  guinea-pigs.  Carbone 
produced  a  fatal  result  in  rabbits  by  intra-venous,  and  in  guinea-pigs  by 

intra-peritoneal  inoculation  ;    the   guinea-pigs 

%^  .      %:.\  *        •.;..  suffered  from  a  purulent  inflammation  of  the 

\:  •,"*•  tunica  vaginalis  accompanied   by  atrophy  of 

:-V-:-        .    *'    *\?  thetestes. 


.....  •"•.•*,•    •        £  SECTION  II.—  MORPHOLOGY. 

:.-       £»**  1.  Microscopical  appearance. 

•&  .\£:.  %.%  The  Micrococcus  melitensis  is  a  rounded  or 

V"   ./£*•  -'        slightly  oval  bacterium,  measuring  about  O3//, 

•]•••         ..       J...  i*1  diameter;  elongated  forms  are  occasionally 

.       "•"  seen    in    cultures.     The   organisms    generally 

no.  -ZM.~  Micrococcus  melitensis.  °ccur  singly  or  as  diplococci  but  may  also  form 

Film  from  an  agar  culture  (24  hours),  very  short  chains.     The  coccus  is  regarded  as 

Carbol^th  3nin.     (Oc.   II,    obj.    Atfc,    non.motilej  though    pollaci    affirmg    ^    it    ig 

motile  and  that  it  has  a  single  flagellum  which 

however  is  very  difficult  to  stain.     Gordon  claims  to  have  demonstrated  one 
to  four  flagella. 

Staining  reactions.—  The  coccus  stains  readily  with  the  ordinary  dyes  and 
is  gram-negative. 

2.  Cultural  characteristics. 

Conditions  of  growth.  —  The  Micrococcus  melitensis  is  an  aerobic  organism. 
The  optimum  temperature  of  growth  is  37°  C.  :    at  22°  C.  the  growth  is 


BIOLOGICAL  PROPERTIES  477 

insignificant.  The  best  medium  for  cultivation  is  5  per  cent,  glycerin- 
agar  but  even  under  the  most  favourable  conditions  growth  is  always  scanty. 

Broth. — Cultivation  of  the  micrococcus  in  broth  gives  rise,  after  incubating 
for  three  days  at  37°  C.,  to  an  uniform  cloudiness  in  the  medium  without  any 
surface  pellicle. 

Agar.  Stab  culture. — Small  spherical  colonies  develop  along  the  line  of 
sowing  and  these  may  ultimately  unite  together  to  form  a  yellowish  streak 
with  denticulated  edges. 

Stroke  culture. — Very  small  transparent  colonies  measuring  2-3  mm.  in 
diameter  are  visible  about  the  third  day  :  on  further  incubation  they  become 
raised,  smooth,  shiny  ana  milky-white  in  appearance. 

On  glycerin-agar  and  glucose-nutrose-agar  the  growth  is  more  rapid  and 
more  abundant. 

Gelatin. — At  22°  C.  the  amount  of  growth  is  nil  or  insignificant.  The 
medium  is  not  liquefied. 

Potato. — No  apparent  growth  takes  place  on  potato. 

Milk. — The  reaction  becomes  alkaline.     The  milk  is  not  coagulated. 


SECTION  in.— BIOLOGICAL  PROPERTIES. 
1.  Vitality. 

Cultures  of  the  micrococcus  will  keep  alive  for  a  long  time  in  the  laboratory  ; 
Shaw  was  able  to  obtain  sub-cultures  from  a  broth  culture  5  months  old  and 
also  from  a  nine -months  old  growth  on  a  dried-up  tube  of  agar. 

In  sterilized  earth  the  organism  lives  at  least  69  days  (Horrocks)  and  in 
cloth  eighty  days.  In  water  and  in  moist  soil  it  does  not  seem  to  live  so  long : 
Horrocks  could  not  recover  the  organism  from  sterile  water  after  a  week  but 
Shaw  in  a  similar  experiment  recovered  it  after  50  days. 

Cultures  can  be  sterilized  by  heating  them  at  60°  or  65°  C.  for  half  an  hour. 

2.  Bio-chemical  reactions. 

The  Micrococcus  melitensis  does  not  ferment  sugars  and  produces  no 
indol. 

3.  Toxin. 

The  toxin  of  the  Micrococcus  melitensis  was  studied  by  Shaw. 

In  monkeys  the  inoculation  of  porcelain-filtered  broth  cultures  only  pro- 
duces a  negligible  reaction.  The  blood  of  the  inoculated  monkeys  exhibits 
feeble  agglutinating  properties  (1  in  80). 

Inoculation  of  cultures  heated  to  60°  or  70°  C.  for  half  an  hour  produces 
hardly  any  more  reaction,  but  the  serum  of  the  inoculated  animal  has  more 
marked  agglutinating  properties  (up  to  1  in  500). 

4.  Immunity.    Vaccination. 

Bruce  has  shown  that  an  attack  of  Mediterranean  fever  renders  the  patient 
immune  to  subsequent  infection,  but  that  the  immunity  is  not  absolute. 
In  monkeys  which  had  recovered  from  one  attack  of  the  experimentally  induced 
disease,  a  second  mild  attack  unaccompanied  by  bacillsemia  was  produced 
by  inoculating  them  a  second  time  (Shaw). 

Animals  easily  resist  the  inoculation  of  large  quantities  of  killed  cultures, 
but  this  does  not  produce  any  immunity  against  the  living  organism,  since 
the  subsequent  inoculation  of  a  small  dose  of  a  living  culture  almost  certainly 
kills  them  (Eyre).  Shaw,  however,  after  giving  monkeys  several  sub- 


478  THE   COCCUS   OF  MEDITERRANEAN   FEVER 

cutaneous  inoculations  of  heated  agar  cultures  found  that  he  could  then 
inoculate  them  with  virulent  cultures  without  producing  a  typical  attack  of 
Mediterranean  fever ;  but  one  of  these  monkeys  showed  no  immunity  to  a 
second  test  inoculation. 

By  repeatedly  inoculating  horses  and  goats  sub-cutaneously  with  living 
cultures  Shaw  and  Eyre  obtained  powerfully  agglutinating  serums  (1  in  3000 
and  1  in  5000).  These  serums  have  no  therapeutic  properties  when  tested 
on  man  and  animals.  In  a  case  of  laboratory  infection  in  man  Nicolle  found 
that  recovery  coincided  with  the  inoculation  of  10  c.c.  of  serum  from  an 
hyper-immunized  ass. 

Bassett-Smith  attempted  the  treatment  of  Mediterranean  fever  with  a 
vaccine  prepared  by  heating  emulsions  of  ten-day  old  agar  cultures  in  distilled 
water  for  half  an  hour  to  60°  C.  The  dose  of  vaccine  used  was  O'5-l  c.c. 
In  acute  cases  the  inoculation  aggravated  the  symptoms  but  in  chronic  cases 
it  appeared  to  stimulate  the  destruction  of  the  micro-organisms  and  certainly 
shortened  the  duration  of  the  disease. 

5.  Agglutination. 

Wright,  Birt  and  Lamb  have  shown  that  the  serum  of  persons  suffering 
from  Mediterranean  fever,  like  the  serum  of  immunized  animals,  agglutinates 
the  micrococcus. 

Generally  speaking  the  agglutination  reaction  is  poorly  developed  in  the  blood 
of  patients  (1  in  15  to  1  in  50),  though  Lamb  and  Kesava  have  obtained  agglutina- 
sion  in  dilutions  of  1  in  160  and  even  1  in  280.  For  purposes  of  clinical  diagnosis 
the  reaction  of  a  1  in  10  or  1  in  15  dilution  of  the  serum  should  be  determined  and 
if  this  gives  a  positive  result  higher  dilutions  may  be  tested.  The  agglutination 
reaction  always  appears  at  the  end  of  the  first  week  of  the  disease,  and  may  still 
be  present  years  after  recovery.  In  artificially  infected  monkeys  Birt  and  Lamb 
have  found  it  present  as  early  as  the  fifth  day. 

The  blood  and  the  milk  of  infected  goats  agglutinate  the  coccus. 

For  carrying  out  the  agglutination  reaction  Nicolle  advises  using  broth  emulsions 
of  agar  cultures  3-5  days  old,  and  mixing  the  serum  and  emulsion  in  small  straight 
tubes.  The  reaction  can  be  observed  with  the  naked  eye. 

Pollaci  and  Ceranlo  have  shown  that  blister  fluid  and  the  saliva  of  infected  persons 
agglutinate  the  organism.  (Dilute  a  loopful  of  an  agar  culture  in  5-20  drops  of 
filtered  saliva  :  the  agglutination  can  be  seen  under  the  microscope  in  30—60  minutes.) 
The  reaction  with  the  saliva  is  said  to  be  always  present  in  persons  suffering  from 
Mediterranean  fever  and  absent  in  healthy  individuals. 

6.  Immune  body. 

Sicre  has  demonstrated  the  presence  of  an  immune  body  in  the  blood  of 
inoculated  animals  and  of  persons  suffering  from  Mediterranean  fever. 


SECTION  IV,— DETECTION,  ISOLATION  AND  IDENTIFICATION 
OF  THE  ORGANISM. 

Post  mortem,  the  spleen,  liver  and  kidneys  should  be  examined  for  the 
Micrococcus  melitensis.  During  life  it  may  be  recovered  by  puncture  of  the 
spleen  (p.  198)  or  by  sowing  cultures  with  the  blood,  milk  or  urine. 

Scrapings  of  the  internal  organs  should  be  sown  on  ordinary  or  litmus- 
nutrose  agar,  incubated  at  37°  C.  for  about  a  week  and  then  examined  and 
tested. 

The  number  of  organisms  in  the  blood  is  always  small :  it  is  best  to  examine 
the  blood  during  the  height  of  the  fever  and  to  sow  at  least  2-4  c.c.  in  250  c.c. 
of  broth.  Pollaci  recommends  the  addition  of  bile  to  the  broth. 


ISOLATION   OF  THE  COCCUS  479 

The  identification  of  the  micrococcus  is  based  upon  the  following  charac- 
teristics : — 

1.  Microscopically,  a  gram-negative,  non-motile  coccus. 

2.  The  rapidity  with  which  a  trace  of  an  agar  culture  breaks  up  in  a  drop 
of  water. 

3.  Absence  of  fermentation  in  sugars,  non-coagulation  of  milk,  and  an 
alkaline  reaction  in  litmus  milk. 

4.  Agglutination  with  a  specific  serum.     The  serum  reaction  (vide  ante) 
will  be  found  of  much  use  in  the  diagnosis  of  Mediterranean  fever. 


CHAPTER  XXXI. 
BACILLUS  MALLEI.1 

Introduction. 

Section  I. — Experimental  inoculation,  p.  480. 

Section  II. — Morphology,  p.  482. 

Section  III. — Biological  properties,  p.  484. 

1.  Vitality  and  virulence,  p.  484.     2.  Toxin  ;  preparation  of  mallein  ;  mallein  in 
the  diagnosis  of  glanders,  p.  484.     3.  Vaccination,  p.  485.     4.  Agglutination,  p.  486. 
Section  IV. — Detection  and  isolation  of  the  bacillus,  p.  486. 

THE  bacillus  of  glanders  was  discovered  independently  by  Lreffler  and 
Schutz  and  by  Bouchard,  Capitan  and  Charrin. 

Glanders  is  almost  entirely  restricted  to  the  Solipedes  though  men  having 
to  do  with  horses  occasionally  contract  the  disease  from  infected  animals, 
and  a  few  cases  are  on  record  in  which  infection  followed  manipulation  of 
cultures  of  the  organism  in  the  laboratory.  The  disease  has  also  been  noticed 
to  occur  spontaneously  among  the  carnivora — lions  and  tigers — after  these 
animals  had  been  fed  upon  meat  from  glandered  animals. 

Two  clinical  types  of  the  disease  are  recognized  depending  upon  whether  the 
lesions  are  more  prominent  in  the  skin — -farcy — or  in  the  internal  organs — glanders 
proper.  The  latter  is  the  more  common  type  :  it  is  characterized  at  the  outset  by 
infection  of  the  nasal  mucous  membrane  and  related  lymphatic  glands,  and  later 
by  lesions  in  the  internal  organs  more  especially  in  the  lungs  and  in  the  genital 
organs  :  the  disease  may  run  either  an  acute  or  chronic  course.  In  farcy,  which 
also  may  assume  an  acute  or  a  chronic  form,  the  chief  lesions  are  abscesses  in  the 
skin — the  so-called  farcy  buds,  which  terminate  in  ulcers — accompanied  by  lym- 
phangitis [farcy  pipes]  and  occasionally  orchitis. 

Glanders  must  be  carefully  distinguished  from  bovine  farcy,  an  entirely  different 
disease,  not  transmissible  to  man  and  due  to  infection  with  a  fungus  of  the  genus 
Discomyces  (Chap.  XLVIII). 


SECTION  I.— EXPERIMENTAL  INOCULATION. 

. — The  ass  is  more  susceptible  to  glanders  than  any  other  animal  and 
inoculation  is  practically  always  followed  by  an  acute  attack  of  the  disease 
though  Arloing  has  recorded  one  instance  in  which  a  chronic  form  of  the 
disease  developed. 

Experimentally  infection  is  usually  produced  by  rubbing  infected  material 
(pus  or  catarrhal  discharge  from  the  nose)  into  a  few  scarifications  made  on 
the  skin  of  the  forehead.  An  oedematous  swelling  rapidly  appears  followed  by 

1  See  footnote  p.  245. 


EXPERIMENTAL  INOCULATION  481 

ulceration  along  the  lines  of  the  scratches  ;  the  temperature  rises  to  40°-41°  G., 
the  neighbouring  glands  become  enlarged,  there  is  a  discharge  from  the  nose 
and  the  animal  dies  in  a  few  days. 

Post  mortem  there  are  nodules  on  the  nasal  and  laryngo-tracheal  mucous 
membranes  and  small  infarcts  in  the  lungs  which  on  pressure  exude  drops  of 
a  thick  very  virulent  pus.  Similar  infarcts  may  be  found  in  the  liver,  kidneys, 
spleen  and  other  internal  organs. 

Mules.  Horses. — Cutaneous  inoculation  in  these  species  is  generally 
followed  by  a  sub-acute  or  chronic  attack  of  glanders.  The  temperature 
may  be  slightly  raised  or  may  remain  normal ;  there  is  a  discharge  from  the 
nose  and  the  glands  in  the  neck  become  enlarged  ;  occasionally  rales  may 
be  heard  in  the  lungs  and  the  animal  may  be  short  of  breath.  In  some 
cases  however  there  may  be  practically  no  symptoms  for  a  long  time.  Post 
mortem  examination  reveals  small  grey  tubercles  in  the  lungs  surrounded  by  a 
narrow  zone  of  congestion.  These  tubercles  consist  of  a  fibrous  shell  containing 
a  small  drop  of  pus. 

Guinea-pigs. — The  guinea-pig  is  nearly  as  susceptible  to  glanders  as  the  ass. 

The  inoculation  of  material  containing  only  the  glanders  bacillus  into  the  peri- 
toneal cavity  of  a  guinea-pig  gives  rise  to  very  characteristic  lesions  (vide  post),  but 
if  other  organisms  be  present  as  well  as  the  glanders  bacillus  the  animal  suffers  from 
an  ordinary  peritonitis.  When  dealing  with  impure  material  therefore  it  is  better  first 
to  isolate  the  organism  in  pure  culture,  which  may  be  done  as  follows :  inoculate 
a  guinea-pig  sub-cutaneously  with  the  material :  an  abscess  will  form  at  the  site 
of  inoculation  and  the  neighbouring  glands  will  become  enlarged.  Excise  one  of 
these  glands,  grind  it  up  in  a  mortar  and  inoculate  the  emulsion  into  the  peritoneal 
cavity  of  a  second  guinea-pig. 

Cutaneous  and  sub-cutaneous  inoculation. — Cutaneous  inoculation  should 
be  done  on  the  back,  and  sub-cutaneous  inoculation  beneath  the  skin  at  the 
top  of  the  thigh.  In  the  former  case  an  ulcer  develops  at  the  site  of  inocula- 
tion, and  in  the  latter  case  a  local  abscess  forms  accompanied  by  lymphan- 
gitis and  swelling  of  the  neighbouring  glands  which  may  break  down  and 
form  abscesses.  The  animal  sickens  and  dies  in  4r-8  weeks. 

It  is  characteristic  of  glanders  that  an  enlargement  of  the  testicle — a 
glanders  sarcocele — often  results  after  inoculation  of  a  male  guinea-pig  : 
about  the  second  week  the  testicles  may  have  reached  a  considerable  size  ; 
the  scrotum  at  first  red  and  tender  soon  begins  to  ulcerate  and  small 
"  chancres  "  are  developed  ;  the  tunica  vaginalis  is  involved,  in  the  early 
stages  it  becomes  adherent  to  the  testicle  and  is  subsequently  infiltrated  with 
small  miliary  abscesses. 

The  lungs,  liver,  spleen  and  lymphatic  glands  are  all  more  or  less  infiltrated 
with  small  miliary  tubercles  with  purulent  centres. 

Intra-peritoneal  inoculation. — A  male  animal  should  be  selected  for  the 
purpose.  The  characteristic  lesion  then  is  the  appearance  of  a  glanders 
sarcocele  after  2  or  3  days  ;  the  animal  generally  dies  during  the  second 
week  after  inoculation.  When  the  inoculated  virus  is  highly  virulent  (cultures, 
for  example)  or  when  the  dose  inoculated  is  large,  death  may  take  place  in  2  or 
3  days  from  septicaemia  before  any  nodular  lesions  have  had  time  to  appear. 

Mice.— Field  mice  are  highly  susceptible  to  glanders  and  succumb  within 
a  week  of  being  inoculated.  The  internal  organs,  and  particularly  the  spleen, 
are  thickly  covered  with  tubercles. 

White  mice  on  the  other  hand  are  more  highly  immune  but  succumb 
after  the  inoculation  of  a  virus  of  increased  virulence. 

Leo  succeeded  in  rendering  white  mice  susceptible  to  glanders  by  feeding  them 
on  phloridzin.  After  being  fed  exclusively  on  biscuits  soaked  in  an  alcoholic 

2n 


482  THE   GLANDERS   BACILLUS 

solution  of  phloridzin  and  dried,  the  mice  became  diabetic  and  then  readily  suc- 
cumbed to  an  inoculation  of  the  glanders  bacillus. 

Ground  squirrels. — Ground  squirrels  are  highly  susceptible  to  glanders  and 
succumb  within  a  week,  the  bacillus  being  distributed  throughout  the  internal 
organs.  The  virulence  of  the  organism  can  be  increased  by  passage  through 
these  animals  (Gamaleia). 

Cats. — Cats  are  susceptible  to  glanders.  Cutaneous  inoculation  is  followed 
by  a  "  chancre,"  death  taking  place  in  15-30  days.  Post  mortem  the  internal 
organs  are  seen  to  be  sprinkled  with  glanders  nodules. 

Sheep.  Goats. — Both  sheep  and  goats  can  be  readily  infected  experi- 
mentally. 

Dogs. — Dogs  are  more  or  less  immune.  In  young  dogs  only  does  the 
disease  become  generalized  and  prove  rapidly  fatal.  Inoculation  of  adults 
of  the  species  through  superficial  skin  scratches  is  followed  by  a  characteristic 
local  lesion.  If  the  inoculation  be  made  on  the  skin  of  the  forehead  the 
part  becomes  oedematous  in  3-5  days  and  ulcers  are  formed  which  exude  a 
very  virulent  discharge.  The  ulcers  extend  for  the  first  week  or  two  then 
become  stationary  and  finally  cicatrize,  the  animal  recovering  completely. 
Nocard  has  however  recorded  fatal  cases  of  the  chronic  form  of  the  disease 
in  dogs. 

The  natural  immunity  of  the  dog  has  been  experimentally  overcome  in  several 
different  ways.  Trasbot,  for  instance,  produced  a  fatal  infection  by  inoculating 
dogs  with  material  from  an  infected  lion.  Strauss  inoculated  huge  doses  of  culture 
into  the  veins  of  adult  dogs  with  the  result  that  the  animals  died  with  lesions  of 
glanders  in  the  skin  and  internal  organs.  Tedeschi  also  induced  a  fatal  infection 
by  inoculating  cultures  into  the  brain,  spinal  cord  and  nerves. 

Rabbits. — Rabbits  are  only  slightly  susceptible  to  experimental  inoculation. 
Sub-cutaneous  inoculation  is  followed  by  an  ulcer  which  resolves  spontaneously. 
Intra-venous  inoculation  of  cultures  is  followed  by  death  (Loe  filer).  A  virus 
which  has  been  passed  through  ground  squirrels  wrill  kill  rabbits  on  sub- 
cutaneous inoculation  (Gamaleia). 

Cattle.  Swine. — These  animals  are  practically  immune  against  glanders. 
Spinola,  however,  has  succeeded  in  infecting  pigs,  and  Cadeac  and  Mallet 
have  shown  that  pigs  are  susceptible  to  infection  when  their  resistance  has 
been  lowered  by  some  antecedent  disease. 

Rats.     Birds. — Both  rats  and  birds  are  immune  to  glanders. 


SECTION   II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  glanders  bacillus  is  a  small,  straight  or  slightly  curved,  non-motile, 
rod-shaped  organism  of  about  the  same  length  (3-5/>0  as  but  thicker  than  the 
tubercle  bacillus  :  the  ends  of  the  bacillus  are  rounded.  In  cultures  the 
organisms  occur  singly  or  in  pairs  while  in  the  tissues  and  in  pus  they  are  often 
found  in  small  masses.  Occasionally  the  bacilli  are  so  short  as  to  have  the 
appearance  of  micrococci,  but  on  the  other  hand  long  branched  filamentous 
forms  are  sometimes  found.  In  old  cultures  involution  forms  consisting 
of  filamentous  irregularly  swollen  bacilli  and  granules  arranged  in  chains  like 
cocci  are  seen. 

Staining  reactions. — The  glanders  bacillus  stains  with  solutions  of  the 
aniline  dyes  containing  a  mordant  such  as  Loeffler's  blue,  Kiihne's  blue, 
carbol-thionin,  or  carbol-fuchsin.  It  does  not  stain  by  Gram's  method. 

In  stained  preparations  the  glanders  bacillus  has  a  granular  appearance, 


MORPHOLOGY  483 

parts  of  the  protoplasm  remaining  unstained  :    these  unstained  parts  do  not 
however  represent  spores. 

Sections.  —  For  staining  the  bacilli  in  sections  either  Nicolle's  tannin  method 
or  one  of  the  following  may  be  employed  : 

Kuhne's  method.  —  1.  On  taking  the  sections 
out  of  alcohol,  wash  them  in  water  and  stain 
for  a  few  minutes  with  carbol-blue. 

2.  Pass  the  sections  rapidly  through  a  1  per 
cent,  aqueous  solution  of  hydrochloric  acid  and 
wash  in  water. 

3.  Dehydrate  very  quickly  in  alcohol  and 
aniline  oil  :  wash  carefully  in  xylol  and  mount 
in  balsam. 

Loeffler's  method.—  1.  Stain  for  a  few  minutes 
in  aniline-fuchsin  (prepared  in  a  similar  manner 
to  aniline-violet)  to  which  1  part  in  10,000  of 

,  .  -,    i         i  111  FIG.   235.  —  Film  preparation  from 

Caustic  potash  has  been  added.  an  infected  testicle  showing  glanders 


2.  Wash  rapidly  in  a  1  per  cent,  solution  of    ^f1-1'    Carb°i-\hionin-   (Reich  ;obj. 

.  .  -.        41T     -,     .  Taiu  m.  ,  oc.  iv.; 

acetic  acid.     Wash  in  water. 

3.  Dehydrate  rapidly  in  alcohol  and  aniline  oil,  wash  carefully  in  xylol 
and  mount  in  balsam. 

2.  Cultural  characteristics. 

Conditions  of  growth.  —  The  glanders  bacillus  is  an  aerobic  organism  ;  it 
hardly  grows  at  all  below  25°  C.,  but  on  glycerin-agar  it  yields  a  scanty 
growth  when  incubated  at  23°-24°  C.  Growth  is  arrested  at  42°  C.  The 
optimum  temperature  for  cultivation  is  35°-38°  C. 

Characters  of  growth  on  ordinary  media.  Broth.  —  When  sown  in  broth 
and  incubated  at  37°  C.  for  24  hours  the  bacillus  produces  first  a  cloudiness  of 
the  medium  and  later  a  white,  mucous  deposit.  A  culture  on  this  medium 
has  no  characteristic  feature. 

Agar.  Glycerin-agar.  —  After  incubating  for  about  24  hours  a  narrow 
whitish  streak  is  seen  along  the  line  of  sowing.  The  culture  is  at  first  semi- 
transparent  but  as  the  layer  thickens  it  becomes  opaque.  On  glycerin- 
agar  growth  takes  place  more  freely  and  may  spread  over  the  whole  surface 
of  the  medium. 

Coagulated  serum.  —  Horse  serum  is  the  best  for  the  growth  of  the  glanders 

bacillus.  Semi-transparent  colonies  appear  in 
about  a  couple  of  days  which  become  white 
and  opaque  as  growth  progresses. 

Gelatin.  —  On  a  12  or  15  per  cent,  gelatin 
—  which  will  remain  solid  at  25°  C.  —  a  very 
scanty  almost  invisible  growth  is  formed  after 
incubating  at  25°  C.  for  several  days. 

Potato.  —  Under  suitable  conditions  the 
§rowth  of  the  glanders  bacillus  on  potato  is 
characteristic.  Potatoes  which  are  either 
naturally  rich  in  starch  or  which  have  been  made  alkaline  should  be  used  if  the 
characteristic  appearances  are  to  be  developed  to  the  best  advantage.  After 
incubating  for  48  hours  at  37°  C.  a  thick  yellowish  viscous  film  appears 
along  the  line  of  sowing,  which  as  the  growth  extends  during  the  next  few 
days  becomes  brown  and  then  acquires  a  chocolate  colour,  while  the  potato 
in  the  neighbourhood  of  the  growth  turns  black. 
Milk.  —  Milk  is  coagulated  in  about  10-12  days. 


484  THE   GLANDERS   BACILLUS 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Viability  and  Virulence. 

Viability. — The  glanders  bacillus  is  a  delicate  organism.  Cultivations  of 
the  bacillus  kept  at  37°  C.  die  out  in  abcnt  a  month,  and  exposure  to  a  tem- 
perature of  55°-60°  C.  will  sterilize  them  in  a  few  minutes.  Bacilli  in  pus 
are  rapidly  killed  by  desiccation,  and  if  some  glanders  pus  be  spread  in  a 
thin  layer  and  left  at  the  ordinary  temperature  of  the  atmosphere  for  48  hours 
it  will  no  longer  produce  an  infection  on  inoculation.  In  the  tissues  and 
internal  organs  the  bacillus  is  more  resistant  but  can  be  destroyed  by  exposure 
to  a  temperature  of  100°  C.  for  a  few  minutes. 

The  glanders  bacillus  is  also  readily  killed  by  antiseptics  so  that  an  exposure 
to  any  of  the  following  solutions  for  a  few  minutes  will  sterilize  it — O'l  per 
cent,  acid  solution  of  perchloride  of  mercury,  3  or  4  per  cent,  solution  of 
cresol,  or  solutions  of  carbolic  acid. 

Virulence. — The  virulence  of  a  given  culture  of  the  bacillus  is  said  to 
disappear  in  a  week.  If  frequently  sub-cultivated  on  artificial  media  the 
virulence  will  be  found  to  have  considerably  diminished  by  the  fifth  or  sixth 
sub-culture.  Young  cultures  of  recently  isolated  organisms  are  very  virulent 
and  a  certain  amount  of  risk  attaches  to  the  manipulation  of  them. 

The  virulence  of  the  bacillus  is  readily  increased  by  passage  through 
certain  animals. 

Gamaleia  raised  the  virulence  of  the  bacillus  considerably  by  passage  through 
ground  squirrels.  Protopopoff  exalted  the  virulence  by  passage  through  rabbits 
and  found  that  after  several  passages  the  virulence  became  fixed,  so  that  on  inocula- 
tion beneath  the  skin  rabbits  invariably  died  in  from  5-8  days.  Trasbot  has  brought 
forward  certain  facts  which  seem  to  show  that  the  virulence  is  increased  by  passage 
through  lions. 

2.  Toxin. 

Cultures  of  virulent  strains  of  the  glanders  bacillus  sterilized  at  100°  C. 
exhibit  toxic  properties  and  quickly  kill  the  inoculated  animal.  The  toxin 
of  glanders  has  not  been  isolated,  but  was  made  the  subject  of  study  first  by 
Kalning  and  Helman  and  afterwards  by  Protopopoff,  by  Roux  and  Nocard, 
and  by  others.  The  term  mallein  has  been  applied  to  an  extract  of  glycerin- 
broth  cultures  of  the  bacillus. 

Preparation  of  mallein  (Nocard). — For  the  preparation  of  mallein  an 
organism  the  virulence  of  which  has  been  raised  and  fixed  by  passage  through 
rabbits  (intra-venous  inoculation)  should  be  used.  Sow  a  glycerin-broth 
medium  with  the  infected  rabbit's  blood  and  incubate  at  36°  C.  for  one 
month ;  then  sterilize  the  culture  by  heating  it  at  100°  C.  for  half  an  hour, 
evaporate  on  a  water  bath  to  one-tenth  its  original  volume  and  filter  through 
Chardin  paper.  The  brown  syrupy  filtrate  constitutes  crude  mallein,  1  c.c. 
of  which  is  sufficient  to  kill  a 'rabbit. 

If  the  crude  product  be  treated  with  several  volumes  of  alcohol  a  preci- 
pitate is  thrown  down  containing  the  active  principle  mixed  with  other 
substances  (dry  mallein  of  Foth). 

Mallein  in  the  diagnosis  of  glanders  (Nocard).— Nocard  was  able  to  demon- 
strate a  very  peculiar  property  of  mallein.  If  inoculated  in  a  very  small 
dose  into  an  healthy  animal  it  leads  to  no  symptoms,  but  if  the  same  dose  be 
administered  to  an  animal  suffering  from  glanders  a  sharp  reaction  is  pro- 
duced similar  to  the  reaction  following  the  inoculation  of  tuberculin  into  a 
tuberculous  animal.  If  O25  c.c.  of  mallein  be  inoculated  into  an  healthy 
horse  no  effect  is  produced,  but  if  a  similar  inoculation  be  given  to  a  glandered 


MALLEIN  485 

horse  a  violent  reaction  follows  characterized  by  an  oedema  at  the  site  of 
inoculation,  rigors,  and  a  rise  of  temperature — of  perhaps  3°  or  4°  C.  within 
24  hours — commencing  a  few  hours  after  the  inoculation  and  persisting  for 
several  days.  Whenever  an  animal  reacts  in  this  way  to  an  inoculation  of 
mallein  a  diagnosis  of  glanders  may  be  made  with  confidence. 

The  inoculation  of  mallein  is  a  valuable  agent  in  the  diagnosis  of  those 
cases  of  glanders  in  the  horse  where  there  is  neither  ulcer  nor  nasal  discharge 
—latent  glanders  ;  but  it  is  not  applicable  to  the  diagnosis  of  glanders  in 
man  on  account  of  the  intensity  of  the  resulting  reaction. 

When  an  animal  is  suffering  from  very  advanced  lesions  or  when  the 
temperature  of  a  suspected  horse  reaches  or  exceeds  39°  C.  the  reaction  may 
fail. 

If  the  inoculation  be  followed  by  a  rise  of  temperature  not  exceeding  1°  or 
1'5°  C.  the  diagnosis  must  be  regarded  as  doubtful,  and  the  animal  should  be 
left  alone  for  3  or  4  weeks  and  then  tested  again  :  or  if  delay  be  inconvenient, 
by  combining  the  original  mallein  test  with  cultivation  and  inoculation 
experiments — carried  out  with  material  from  the  suspected  animal — it 
should  be  possible  to  come  to  a  definite  conclusion. 

The  method  of  conducting  the  mallein  test. — In  veterinary  practice  a  diluted 
solution  of  mallein  should  be  used  rather  than  the  crude  product. 

5  per  cent,  aqueous  solution  of  carbolic  acid.         -  9  paits. 

Crude  mallein,  -  1  part 

The  horse  to  be  tested  should  be  kept  in  its  stable  for  2  days  before  the 
test  is  performed  and  its  temperature  taken  morning  and  evening — because 
as  already  pointed  out  if  the  temperature  before  inoculation  exceed  39°  C. 
the  reaction  may  fail.  On  the  third  day  2*5  c.c.  of  the  diluted  mallein  is 
inoculated  beneath  the  skin  of  the  shoulder  and  the  temperature  taken  thrice 
daily.  In  animals  infected  with  glanders  the  temperature  will  begin  to  rise 
about  8-10  hours  after  the  inoculation  and  will  remain  up  for  about  2  days. 

Experiments  have  been  carried  out  to  determine  whether,  as  is  the  case  in 
the  diagnosis  of  tuberculosis  with  tuberculin,  a  characteristic  reaction  occurs 
in  glandered  animals  if  mallein  be  instilled  into  the  eye  or  dropped  on  super- 
ficial skin  scratches  ;  but  all  observers  are  agreed  that  these  methods  of 
applying  the  test  are  neither  so  reliable  nor  so  constant  in  their  results  as  the 
original  method  of  sub-cutaneous  inoculation. 

3.  Vaccination. 

There  are  considerable  difficulties  in  the  way  of  prophylactic  vaccination 
against  glanders  and  up  till  the  present  the  results  have  not  been  at  all 
satisfactory. 

Straus  having  shown  that  dogs  could  be  infected  by  inoculating  cultures  of 
exalted  virulence  into  the  veins  found  that  a  previous  inoculation  of  old 
attenuated  cultures  protected  the  animals  against  infection  by  intra-venous 
inoculation.  But  dogs  immunized  in  this  way  are  not  immune  against 
cutaneous  inoculation  which  is  followed  by  cutaneous  ulcers  ;  and  Galthier  has 
shown  that  by  repeatedly  inoculating  an  animal  ulcers  can  be  produced  on  as 
many  as  five  successive  occasions. 

Sakaroff  and  Finger  found  that  a  previous  inoculation  of  old  cultures  or 
of  cultures  sterilized  by  heat  at  100°  C.  caused  the  disease  to  run  a  slower 
course  than  usual  in  rabbits  but  they  were  not  able  to  prevent  the  animals 
from  dying. 

Nicolle  has  attempted  the  immunization  of  guinea-pigs  by  intra-cardiac 
inoculation  of  a  virulent  virus. 

The  immunizing  experiments  of  Babes  (injections  of  mallein).  of  Sakaroff 


486  THE   GLANDERS   BACILLUS 

(inoculation  of  horses  with  a  bacillus  after  passage  through  cats),  and  of 
Chenot  and  Picq  (inoculation  of  guinea-pigs  with  ox  serum)  have  given  no 
conclusive  results. 

4.  Agglutination. 

In  testing  the  agglutination  reaction  of  the  serum  of  an  animal  infected 
with  glanders  an  emulsion  in  normal  saline  solution  of  a  young  culture  on 
glycerin-agar  should  be  used.  It  is  better  to  sterilize  the  culture  by  heating 
it  at  60°  C.  for  an  hour. 

The  serum  of  healthy  horses  agglutinates  the  bacillus  in  dilutions  of  1  in  100 
to  1  in  300,  while  that  of  infected  animals  under  the  same  conditions  will 
agglutinate  the  bacillus  in  dilutions  of  1  in  500  to  1  in  1000  (Bourges  and 
Mery,  M'Fadyean,  Pokchichevsky). 

Normal  human  serum  also  agglutinates  the  glanders  bacillus  but  the 
reaction  is  more  marked  in  the  case  of  persons  suffering  from  the  disease. 

On  the  whole  the  agglutination  reaction  does  not  afford  a  practical  method 
for  the  diagnosis  of  glanders. 

SECTION  IV.— DETECTION  AND   ISOLATION  OF  THE   GLANDERS 

BACILLUS. 

Distribution  of  the  bacillus  in  the  tissues. — In  cases  of  glanders  the  bacillus 
can  be  found  in  the  pus,  in  the  discharges  from  the  ulcers  and  nasal  mucous 
membrane,  in  the  farcy  buds,  and  in  tubercles  and  infarcts. 

The  lymphatic  system  is  the  site  of  election  of  the  bacillus  ;  as  a  rule  the 
lymphatic  glands  are  infected  at  an  early  stage,  but  this  is  not  invariably 
the  case,  and  Nocard  has  shown  that  the  enlarged  glands  in  the  neck  do  not 
always  produce  glanders  on  inoculation  into  suitable  animals. 

In  the  lower  animals  the  bacillus  is  practically  never  found  in  the  blood 
(Nocard)  except  in  very  acute  forms  of  the  disease  (Lixteyn  and  Preusse). 
In  man  the  bacillus  is  found  in  the  blood  more  often  than  in  animals  (Loeffler, 
Goutchakofi,  Sittmann). 

The  saliva,  urine,  secretion  of  the  testicles  and  of  the  sweat  glands  have 
in  some  cases  been  found  to  be  infected  but  in  no  case  has  the  milk  been 
shown  to  contain  the  bacillus. 

Note. — It  is  often  impossible  to  detect  the  glanders  bacillus  by  microscopical 
examination  even  in  films  of  pus  or  of  the  contents  of  the  tubercles  from  cases  of 
glanders.  The  presence  or  absence  of  the  glanders  bacillus  can  neither  be  affirmed 
nor  denied  on  microscopical  examination  alone  ;  cultivation  and  inoculation  experi- 
ments must  be  carried  out  in  every  case.  The  failure  to  find  the  specific  bacillus 
is  particularly  common  in  chronic  lesions,  and  especially  in  lesions  of  the  horse. 
To  find  the  bacillus  by  microscopical  examination  pus  from  dogs,  or  an  enlarged 
testicle  from  a  guinea-pig,  or  material  from  an  acute  lesion  in  the  ass  should  be 
used. 

The  diagnosis  of  glanders. 

The  clinical  diagnosis  of  glanders  is  often  difficult  and  sometimes  impossible 
without  laboratory  methods.  The  early  diagnosis  of  latent  glanders  is  onh 
possible  by  using  mallein  in  the  manner  described  above.  The  present 
section  is  concerned  only  with  laboratory  methods  of  diagnosis — the  detec- 
tion and  isolation  of  the  bacillus. 

1.  Microscopical  examination. — Pus,  discharges  from  sores  and  scrapings 
of  the  internal  organs,  etc.  will  provide  the  material  for  examination.  The 
films  should  be  stained  in  the  manner  already  described.  The  bacillus  is 
gram-negative.  Pieces  of  tissue  for  histological  examination  should  be 
hardened  in  alcohol  and  embedded  in  paraffin. 


DIAGNOSIS   OF  GLANDERS  487 

2.  Cultures. — Pus,  scrapings  of  organs  and  other  material  must  be  collected 
with  the  necessary  aseptic  precautions  and  should  invariably  be  sown  on 
potato.     The  appearance  presented  by  the  glanders  bacillus  on  this  medium 
is  characteristic   (vide  ante)  and  is  an  important  factor  in  the  diagnosis. 
Several  tubes  of  potato  should  be  sown  in  order  to  isolate  the  organism 
which  may  not  always  be  present  in  pure  culture  in  the  material  used. 

3.  Inoculations. — Before  the  discovery  of  mallein  the  inoculation  of  animals 
with  pus,  nasal  discharge  and  other  material  from  suspected  cases  of  glanders 
was  an  experiment  of  first  rate  importance  in  the  diagnosis  of  the  disease. 
It  has  however  been  pointed  out  above  that  enlarged  glands  taken  from 
animals  suffering  from  the  disease  may  fail  to  produce  glanders  on  inoculation 
into  healthy  animals,  and  the  method  is  to  this  extent  a  less  certain  means 
of  diagnosis  than  the  mallein  test :   it  however  affords  valuable  confirmatory 
evidence. 

The  suspected  material  should  be  inoculated  into  a  guinea-pig,  an  ass  or  a  dog. 

(a)  Guinea-pigs. — The  inoculation  of   suspected   material  into  the  peri- 
toneal cavity  of  a  guinea-pig  has  been  recommended  by  Strauss  as  at  once 
the  simplest  and  most  certain  method  of  diagnosing  a  case  of  glanders.     The 
difficulty  however  is  that  the  material  used  for  inoculation  must  contain 
no  organisms  capable  of  setting  up  peritonitis  in  the  inoculated  guinea-pig, 
and  in  practice  it  is  found  that  about  one-half  of  the  animals  inoculated 
with  the  discharge  from  the  nose  die  of  septic  peritonitis  in  24—36  hours. 

If  the  material  therefore  contains  organisms  other  than  the  glanders  bacillus 
it  should  be  inoculated  beneath  the  skin  of  a  guinea-pig,  and  a  second  guinea- 
pig  should  be  inoculated  intra-peritoneally  with  a  portion  of  a  lymphatic 
gland  from  the  first  animal.  (In  these  cases  however  it  is  often  better  to 
inoculate  an  ass  with  the  suspected  material.) 

For  purposes  of  inoculation  rub  up  a  little  of  the  pus  or  nasal  discharge 
or  other  material  in  a  mortar  with  a  little  sterile  water  [or  normal  saline 
solution]  and  inject  the  emulsion  into  the  peritoneal  cavity  of  a  male  guinea- 
pig.  In  2-3  days  the  characteristic  enlargement  of  the  testicle  will  become 
apparent  and  the  animal  will  die  in  a  week  to  a  fortnight. 

For  a  long  time  the  appearance  of  an  enlargement  of  the  testicle — Straus'  sign — 
following  the  inoculation  of  material  from  a  suspected  case  of  glanders  into  the 
peritoneal  cavity  of  a  male  guinea-pig  was  regarded  as  pathognomonic  of  glanders 
and  as  absolute  proof  of  the  material  having  been  derived  from  a  case  of  the  disease. 
But  Kutschen  has  isolated  from  the  nasal  discharge  of  a  glandered  horse  an  organism 
which  while  differing  from  the  glanders  bacillus  in-  other  respects,  on  inoculation 
into  the  peritoneal  cavity  of  a  male  guinea-pig  produces  an  orchitis  similar  to  the 
orchitis  produced  by  the  glanders  bacillus.  Hallopeau  and  Bureau  observed  a 
similar  orchitis  develop  after  inoculating  pus  from  a  case  of  human  mycosis  into  the 
peritoneal  cavity  of  a  guinea-pig.  And  Nocard  has  recorded  nineteen  cases  of  a 
slightly  contagious,  farcy-like  lymphangitis  in  horses  due  to  a  bacillus  which  though 
it  produced  an  orchitis  on  inoculation  into  guinea-pigs  was  absolutely  different  from 
the  glanders  bacillus  both  in  its  cultural  characteristics  and  in  its  reaction  to  Gram's 
stain.  The  inoculation  of  a  guinea-pig  therefore  can  only  be  regarded  as  one  factor 
in  the  diagnosis  of  a  doubtful  case  of  glanders  and  must  be  supplemented  in  every 
case  by  a  microscopical  examination  of  the  pus  in  the  testicle  and  by  the  mallein 
test  (Nocard). 

(b)  Asses. — The  susceptibility  of  the  ass  to  glanders  renders  inoculation 
of  that  animal  a  valuable  means  of  diagnosis :  the  animal  should  be  inoculated 
through  superficial  scratches  on  the  skin.    If  the  material  used  for  inoculation 
contain  the   specific   bacillus  the  animal  will  almost  invariably  show  the 
characteristic  symptoms  of  the  disease  before  the  end  of  the  second  week 
(but  see  p.  480). 


CHAPTER   XXXII. 
VIBRIO  CHOLERA   ASIATICS. 

Introduction 

Section  I. — The  experimental  disease,  p.  489. 

1.  Choleraic  peritonitis,  p.  489.  2.  Choleraic  septicaemia,  p.  489.  3.  Intestinal 
cholera  in  animals  and  man,  p.  489. 

Section  II. — Morphology  and  cultural  characteristics,  p.  491. 

Section  III. — Biological  properties,  p.  493. 

1.  Vitality  and  virulence,  p.  493.  2  Bio-chemical  reactions,  p.  494.  3  Toxin, 
p.  494.  4.  Vaccination,  p.  496.  5.  Serum  therapy,  p.  498.  6.  Bactericidal  pro- 
perties ;  Agglutination,  p.  499.  7.  Complement  fixation,  p.  500. 

Section  IV. — Detection,  isolation  and  identification  of  the  vibrio,  p.  500. 
1.  Detection,  p.  500.     2.  Isolation,  p.  501.     3.  Identification,  p.  502. 

The  vibrio  of  Finkler-Prior,  p.  502. 
The  vibrio  of  Deneke,  p.  503. 
Vibrio  metchnikowi,  p.  503. 

THE  infecting  agent  in  asiatic  cholera  is  a  vibrio  discovered  by  Koch  and 
often  known  as  the  comma  bacillus.  The  vibrio  is  found  in  the  intestinal 
contents  and  in  the  dejecta  of  patients  suffering  from  the  disease  :  it  remains 
localized  in  the  intestine  and  the  symptoms  of  cholera  are  due  to  the  absorption 
of  toxin. 

[The  present  conception  of  the  distribution  of  the  cholera  vibrio  in  the 
tissues  of  man  must  however  be  revised  in  view  of  the  recent  observations  of 
Kulescha  and  of  Greig.  Kulescha  was  the  first  apparently  to  show  that 
the  cholera  vibrio  could  gain  access  to  the  gall-bladder  and  set  up  patho- 
logical changes  in  the  biliary  passages  and  Greig  was  able  to  isolate  the  vibrio 
from  the  bile  of  about  one-third  (81  out  of  271)  of  the  fatal  cases  of  cholera 
coming  under  his  observation  at  Puri  in  India.  Moreover  the  finding  by 
Zlatogoroff  of  the  vibrio  in  the  stool  of  a  person  one  year  after  recovery 
leads  to  the  suspicion  that  the  organism  may  live  in  the  gall-bladder  for 
long  periods  and  be  excreted  via  the  alimentary  canal  from  time  to  time 
thus  giving  rise  to  "  carriers  "  as  in  the  case  of  enteric  fever  and  other 
diseases.] 

The  cholera  vibrio  is  essentially  a  pleomorphic  organism  ;  there  are  many 
varieties  which  differ  more  or  less  from  the  vibrio  originally  described  by  Koch. 
If  it  be  added  that  it  is  not  uncommon  to  find  in  water  and  in  the  excreta  of 
healthy  persons,  vibrios  morphologically  similar  to  if  not  identical  with  the 
cholera  vibrio,  it  will  be  understood  how  difficult  and  inexpedient  a  diagnosis 
of  cholera  may  be  in  the  absence  of  large  epidemics. 


EXPERIMENTAL  INFECTION  489 


SECTION  I.— THE   EXPERIMENTAL   DISEASE. 

1.  Choleraic  peritonitis. 

The  inoculation  of  a  culture  of  the  cholera  vibrio  into  the  peritoneum  of 
a  guinea-pig  generally  leads  to  a  rapidly  fatal  peritonitis  (Pfeiffer)  ;  but  this 
experimental  peritonitis  has  no  analogy  whatever  with  the  intestinal  disease 
of  man. 

Vibrios  from  different  sources  differ  widely  in  their  virulence  for  the  same 
animal  species.  Thus  some  vibrios  which  are  not  derived  from  cases  of 
cholera  in  man  will  produce  peritonitis  in  guinea-pigs,  while  others  only 
recently  isolated  from  the  intestine  of  a  person  suffering  from  cholera  may 
prove  absolutely  harmless  to  these  animals. 

There  seems  to  be  no  relationship  between  the  power  of  a  vibrio  to  set  up  choleraic 
peritonitis  in  guinea-pigs  and  its  ability  to  produce  intestinal  cholera. 

To  produce  choleraic  peritonitis  in  the  guinea-pig  scrape  the  growth  from  a 
young  agar  culture,  rub  it  up  in  a  little  sterile  broth  and  inject  the, emulsion 
into  the  peritoneal  cavity.  A  few  hours  later  the  animal  becomes  drowsy, 
the  temperature  falls  below  normal,  collapse  sets  in  and  is  followed  by  con- 
vulsions and  death.  Post  mortem  there  is  considerable  fluid  in  the  peritoneal 
cavity  in  which  a  variable  but  small  number  of  vibrios  can  be  found  :  the 
intestine  is  distended  and  pink  in  colour,  and  the  bacillus  will  be  found  in 
small  numbers  in  its  contents  :  there  will  be  no  visible  lesions  of  the  viscera. 
The  vibrio  may  gain  access  to  the  blood  stream. 

The  virulence  of  a  vibrio  may  be  increased  by  passage  through  the  peritoneal 
cavities  of  guinea-pigs,  but  after  passage  through  about  twenty  animals  the 
virulence  of  the  exalted  virus  appears  to  be  fixed  (Haffkine)  and  cannot  be 
further  increased. 

2.  Choleraic  septicaemia. 

Infection  of  guinea-pigs  and  rabbits  by  sub-cutaneous  inoculation  can 
only  be  effected  when  very  virulent  strains  are  used.  In  such  cases  the  animal 
dies  more  or  less  rapidly  from  choleraic  septicaemia,  death  being  preceded  by 
a  fall  of  temperature,  convulsions  and  collapse  :  the  blood  and  viscera  yield 
pure  cultures  of  the  vibrio.  Ground  squirrels  are  much  more  susceptible  to 
inoculation  with  the  cholera  vibrio  than  guinea-pigs. 

Intra-muscular  inoculation  appears  to  produce  more  severe  symptoms 
than  sub-cutaneous  inoculation.  Intra-venous  inoculation  produces  in 
rabbits  symptoms  like  those  of  cholera  with  lesions  in  the  intestines  (bluish 
red  in  colour  with  desquamation  of  the  mucous  membrane),  while  the  organism 
is  found  in  large  numbers  in  the  contents  of  the  intestine,  blood  and  viscera 
(Kolb  and  Issaeff). 

Pigeons. — Non-pathogenicity  for  pigeons  was  for  a  long  time  thought  to  be 
one  of  the  characteristic  features  of  the  cholera  vibrio.  This  however  is  not  the 
fact  for  Gamaleia  and  Metchnikoff  have  shown  that  many  undoubted  cholera 
vibrios  are  pathogenic  for  these  birds  ;  the  Angers  vibrio,  for  instance,  if 
inoculated  into  the  pectoral  muscles  of  pigeons  will  produce  a  rapidly  fatal 
septicaemia. 

3.  Intestinal  cholera. 

The  symptoms  following  sub-cutaneous  or  intra-venous  inoculation  of  the 
cholera  vibrio  are  very  different  from  those  characteristic  of  asiatic  cholera 
in  man.  Attempts  to  infect  animals  by  the  alimentary  canal  failed  to  pro- 
duce satisfactory  results  until  the  subject  was  taken  up  by  Metchnikoff  ;  as 
a  result  of  his  experiments  a  considerable  advance  was  made  in  the  study 
of  experimental  intestinal  cholera. 


490  THE   CHOLERA   VIBRIO 

Animals. 

I.  Animals  suffer  no  harm  from  being  fed  with  cultures  of  the  vibrio  or 
with  cholera  stools  ;   Nicati  and  Rietsch  therefore,  with  the  object  of  inocu- 
lating the  material  directly  into  the  intestine,  injected  cultures  into  the 
duodenum  of  guinea-pigs  after  laparotomy.     These  observers  were  the  first 
to  produce  intestinal  cholera  experimentally. 

II.  Koch  obtained  the  same  results  but  in  another  way.     He  injected 
directly  into  the  stomach  of  a  guinea-pig  through  an  cesophageal  tube,  first 
a  few  c.c.  of  a  2  per  cent,  solution  of  carbonate  of  soda  and  a  few  minutes 
later  a  culture  of  the  vibrio  :    at  the  same  time  tincture  of  opium  (1-1 '5  c.c.) 
was  inoculated  either  into  the  peritoneal  cavity  or  beneath  the  skin  (Doyen 
uses  40  per  cent,  alcohol  instead  of  opium).     The  animal  died  in  two  or  three 
days  from  diarrhoea  and  collapse.  •  Post  mortem,  the  small  intestine  contained 
a  watery  fluid  in  which  cream-coloured  flakes  were  suspended  and  which 
yielded  an  almost  pure  culture  of  the  vibrio. 

III.  Zattolotny  showed  that  ground  squirrels  are  very  susceptible  to  infec- 
tion with  the  cholera  vibrio  :    if  a  number  of  these  animals  be  fed  with 
foodstuffs  watered  with  a  few  drops  of  a  pure  culture  of  the  vibrio,  half  the 
number  become  infected  and  die  ;    the  mortality  is  heavier  if  an  alkaline 
salt  be  added  to  the  infected  meal,  though  some  of  the  animals  even  then  resist 
infection.     The  affected  animals  become  weak  and  frequently  suffer  from 
diarrhoea,  sometimes  also  from  cramp  with  cyanosis  of  the  nose  and  tongue  ; 
the  temperature  is  sub-normal.     Post  mortem  the  intestinal  canal  is  dis- 
tended and  hyperaemic  and  contains  a  fluid  rich  in  vibrios  ;    the  latter  often 
gain  access  to  the  peritoneal  cavity  and  blood  stream. 

IV.  Metchnikoff,  conceiving  the  immunity  of  animals  to  intestinal  cholera 
to  be  largely  due  to  the  action  of  organisms  normally  present  in  the  intestine, 
experimented  with  a  view  to  overcoming  or  at  least  diminishing  such  pro- 
phylactic action  if  it  existed.     He  fed  a  number  of  young  rabbits  solely 
on  their  mothers'  milk  for  some  weeks  :    the  intestinal  flora  under  these 
conditions  remained  for  a  long  time  quite  poor  and  but  little  varied.     The 
growth  on  a  twenty-four-hour  agar  culture  (Massaouah  vibrio  *)  was  scraped 
off  with  the  bent  end  of  a  pipette  and  placed  in  the  mouth  of  the  young 
rabbits  :    in  about  one-half  the  cases  the  animals  suffered  from  diarrhoea 
-and  died  of  intestinal  cholera  about  the  sixth  day.     Post  mortem  the  intes- 
tines showed  the  characteristic  lesions  of  cholera  and  numerous  vibrios  were 
found  in  the  contents  of  the  intestine. 

V.  Young  chimpanzees  can  be  fed  with  large  quantities  of  the  cholera 
vibrio  without  showing  any  symptoms. 

VI.  Ancillary  micro-organisms. — Metchnikoff,  after  showing  that  in  gelatin 
plate  cultures  some  micro-organisms  favour  the  growth  of  the  cholera  vibrio, 
investigated   these  ancillary  properties   particularly  with   regard    to    three 
organisms  isolated  from  the  human  stomach,  viz.  :   a  white  sarcina,  a  torula 
and  a  bacillus  belonging  to  the  colon  group.     Twenty  out  of  twenty-two 
young  rabbits  fed  with  a  mixture  of  the  Massaouah  vibrio  and  these  organisms 
died  of  cholera.     As  a  rule  death  occurred  36-48  hours  after  infection,  but 
in  a  few  cases  was  longer  delayed  ;  nearly  all  the  animals  were  dead  within  60 
hours.     The  infected  animals  suffered  from  a  watery  diarrhoea  with  a  colour- 
less,  serous,   alvine  discharge  containing  lumps  of  mucus  :    vomiting  was 
rare,  but  suppression  of  urine  very  common.     The  abdominal  walls  were 

1 A  vibrio  isolated  from  some  water  at  Versailles  was  found  to  be  virulent  for  guinea- 
pigs  on  intra-peritoneal  inoculation  and  to  have  the  same  effect  on  young  rabbits  as  the 
Massaouah  vibrio. 


MORPHOLOGY  491 

soft  and  flabby,  the  temperature  fell  to  30°  C.  or  below  and  death  took  place, 
occasionally  after  a  very  prolonged  agony.  Post  mortem,  there  were  no 
lesions  of  the  abdominal  or  thoracic  viscera,  the  small  intestine  alone  being 
hypersemic,  pink  in  colour  and  distended  with  a  turbid  fluid  :  the  caecum 
contained  a  large  amount  of  thick  alkaline  serous  fluid  with  mucous  flakes 
in  suspension.  The  fluid  in  the  small  intestine  contained  an  enormous 
number  of  vibrios,  most  frequently  in  pure  culture.  The  micro-organisms 
ingested  at  the  same  time  as  the  vibrios  disappeared  after  performing  their 
ancillary  role.  In  one-fourth  of  the  cases  the  vibrio  passed  into  the  blood 
stream. 

When  young  rabbits  cease  to  feed  entirely  on  milk  their  susceptibility  to 
infection  with  cholera  vanishes,  and  an  immunity  is  established  which  cannot 
be  overcome  even  with  the  assistance  of  other  micro-organisms.  The 
intestinal  cholera  of  young  rabbits  is  contagious  and  may  be  transmitted 
by  the  mammse  of  the  mother  during  suckling  of  the  infected  animals. 

Young  guinea-pigs  a  few  days  old  are  much  less  susceptible  to  infection 
than  rabbits  when  fed  with  a  mixture  of  the  Massaouah  vibrio  and  ancillary 
organisms.  The  disease  from  which  they  suffer  is  less  characteristic  of  cholera 
than  the  disease  in  rabbits,  and  the  vibrio  exhibits  a  greater  tendency  to 
become  generalized  in  the  tissues  of  guinea-pigs. 

Man. 

It  is  many  years  since  experiments  were  first  conducted  with  a  view  to 
infecting  the  human  subject  with  cholera  by  the  alimentary  canal  (Boche- 
fontaine,  Klein).  In  1892,  Pettenkofer  and  Emmerich  swallowed  pure  cul- 
tures of  Koch's  vibrio,  but  though  they  had  previously  taken  carbonate  of 
soda  and  strictly  regulated  their  diet,  they  merely  suffered  from  an  attack 
of  choleraic  diarrhoea  unaccompanied  by  any  general  symptoms. 

Hasterlik  and  Strieker,  and  Ferran  also  suffered  from  diarrhoea  and  vomiting 
after  taking  pure  cultures  of  the  vibrio. 

On  several  occasions  Metchnikoff  and  his  pupils  drank  pure  cultures  of 
vibrios  from  different  sources  (from  Hamburg,  Courbevoie,  Saint-Cloud, 
Paris,  Versailles,  etc.).  The  observer  first  took  a  gram  of  bi-carbonate  of 
soda  dissolved  in  a  little  water  and  immediately  afterwards  a  varying  amount 
of  an  agar  culture  rubbed  up  in  a  little  sterile  broth.  Metchnikoff  was  able 
in  this  way  to  produce  "  a  true  asiatic  cholera  which  although  slight  had 
all  the  classical  symptoms "  of  the  disease : 
'"  rice  water  "  stools,  sub-normal  temperature,  jr 

vomiting,    cramps,    suppression    of   urine,    and          ^  -  ^ 

vibrios  in  almost  pure  culture  in  the  stools.  ^      ^ 

„  _  -       « 

SECTION   H.— MORPHOLOGY.  .         x         o> 

f  -  ^  f* 

Cholera  vibrios  are  essentially  pleomorphic ;  x    f 

their  shape,  the   number    of   their  flagella  and      f  * 

their  cultural  characteristics  being  all  very  vari-         \    I  * 

able.     This  pleomorphism  renders  their  identifi- 
cation  singularly  difficult.  ^t 

FIG.  237. — Vibrio  ckolerce  (Indian 

1.    Microscopical  appearance.  strain).     Film  from  an  agar  culture. 

Dilute  carbol-fuchsin.     (Reich  ;  oc. 

The  typical  cholera  vibrio  (of  Koch)  occurs  as  n. :  obj.  Tuh.) 
a  stumpy  rod — 1'5-3/x,  long  and  0'5-0'6/j  broad — 

slightly  curved  like  a  comma  ;    the  degree  of  curvature  is  very  variable.     In 
the  field  of  the  microscope  some  of  the  vibrios  appear  to  be  straight,  but 


492  THE   CHOLERA  VIBRIO 

in  these  the  line  of  curvature  is  perpendicular  to  the  surface  of  the  slide  : 
the  eye  only  sees  the  projection  on  the  plane  of  the  slide  and  the  curve 
vanishes.  The  vibrio  is  flagellated  and  is  very  motile. 

There  are  however  varieties  of  the  cholera  vibrio  which  differ  markedly  from  that 
of  Koch.  Some  are  slender,  irregularly- curved  and  occasionally  have  an  elongated 
S  shape — the  Massaouah,  Courbevoie  and  Paris  vibrios.  Others  are  straight 
and  never  show  any  curve — the  Shanghai  vibrio  ;  others  again  are  very  small  and 
of  a  cocco-bacillary  form — the  Malta  vibrio.  Metchnikoff  also  noticed  further 
when  sub-cultivating  an  old  culture  of  the  Angers  vibrio  in  peptone  water  that  it 
had  a  slender  elongated  form,  whereas  ordinarily  it  was  stumpy  and  curved. 

Involution  forms  occur  in  cultures  several  days  old  :  many  of  the  organisms 
are  irregularly  swollen  while  others  have  the  form  of  rounded  bacilli  of 
variable  size. 


FIG.  238.— Vibrio  cholerce  (Massaouah  strain).  FIG.    239.— Vibrio   cholerce   (Indian   strain). 

Film  from  intestinal  contents.     Dilute  carbol-  Stained  to  show  flagella.      Nicoile's    method, 

fuchsin.    (Reich  ;  oc.  II. ;  obj.  TUh.)  (Reich  ;  oc.  IV. ;  obj.  TUh.) 

According  to  Hueppe,  some  of  these  spherical  bodies  represent  resistant 
forms  or  arthrospores  formed  by  encystment  of  the  vibrios  ;  they  are  however 
no  more  resistant  to  adverse  influences  than  the  vibrio  itself. 

Staining  reactions. — The  cholera  vibrio  is  not  so  easily  stained  as  most 
pathogenic  bacilli  and  rather  strong  staining  solutions  containing  a  mordant 
should  be  used.  Carbol-fuchsin  diluted  with  3  or  4  times  its  volume  of  water 
is  a  very  useful  stain.  The  vibrios  are  gram-negative. 

Flagella. — The  number  of  flagella  and  their  arrangement  are  very  variable. 
The  typical  cholera  vibrio  of  Koch  has  one  flagellum  situated  terminally 
[monotrichous]  :  some  varieties  have  two,  three  or  even  four  terminally 
situated  flagella  [lophotrichous]  (Nicolle  and  Morax,  Kolle,  and  Gotschlich). 
The  flagella  may  be  stained  in  the  living  condition  by  Straus'  method  or,  after 
drying  and  fixing,  by  the  methods  described  at  p.  148  et  seq. :  it  is  necessary 
always  to  use  young  agar  cultures. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  cholera  vibrio  is  essentially  an  aerobic  organism  : 
a  very  scanty  growth  may  however  be  obtained  under  anaerobic  conditions 
(Hueppe  and  Scholl).  The  organism  grows  at  all  temperatures  between 
12°  and  40°  C.,  the  optimum  being  37°  C.  It  grows  on  all  the  ordinary 
neutral  or  slightly  alkaline  media  and  ferments  sugars. 

Characteristics  of  growth.  Broth.  Peptone  water.— When  sown  in  these 
media  and  incubated  at  37°  C.  the  cholera  vibrio  rapidly  (6-10  hours)  produces 
a  cloudiness  of  the  medium,  and  later  a  thin  whitish  very  delicate  pellicle 
forms  on  the  surface  of  the  liquid  ;  ultimately  a  flaky  precipitate  is  deposited. 


BIOLOGICAL  PROPERTIES 


493 


Gelatin.  Stab  culture. — After  incubating  at  20°  C.  for  20  hours  small 
colonies  appear  along  the  line  of  the  stab.  A  small  cup-shaped  depression 
is  formed  at  the  surface  in  which  a  bubble  of  air  is  retained  ; 
liquefaction  then  increases  and  progresses  in  a  funnel-shaped 
manner  being  more  marked  at  the  surface  than  in  the  depth, 
the  bubble  of  air  remaining  at  the  surface  :  about  the  second 
to  the  fourth  day  the  growth  is  characteristic.  The  medium 
is  subsequently  entirely  liquefied. 

Single  coloni es. —After  incubating  at  20°  C.  for  about  20 
hours  small  whitish  points  are  visible  which  quickly  become 
irregular-shaped  colonies  with  granular  centres  surrounded 
by  a  bright  ring.  Liquefaction  then  commences  :  a  small 
cup-shaped  depression  is  formed  with  the  colony  in  the  centre 
from  the  periphery  of  which  small  clumps  of  vibrios  become 
detached,  and  the  plate  is  soon  completely  liquefied. 

Agar. — Incubation  at  37°  C.  gives  a  copious  whitish  growth 
which  develops  rapidly  but  has  no 
special  features. 

Isolated  colonies  are  irregular  and 
greyish :  the  centres  are  granular 
and  surrounded  by  a  smooth  mar- 
ginal zone. 

Coagulated  serum. — The  growth  on 
this  medium  is  rapid.  The  serum 
is  liquefied. 

Pntatn        Thp  nTinlpra  vihrin  CTTT»W«     strain).  Stab  culture 

roiato. —  LO  grows   in  geiatin  (5  days), 

well    only    on    alkaline   potato    (p. 
55)  ;  on  this  medium  a  thick  clear  brown  streak  is 
formed. 

Milk. — This  medium  is  sometimes  coagulated. 
Koch  believed  that  one  of  the  characteristics  of  the  cholera  vibrio  was  that 
it  did  not  clot  milk.     Since  then  however  it  has  been  shown  that  some 
varieties  of  cholera  vibrios  of  the  identity  of  which  there  can  be  no  doubt 
produce  quite  a  distinct  clot. 


FIG.  240—  Vibrio 
cholerce     (Indian 


FIG.  241. —  Vibrio  cholerw 
(Indian  strain).  Single  colony 
on  gelatin  plate,  x  60. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Vitality  and  Virulence. 

The  cholera  vibrio  retains  its  vitality  for  a  long  time  in  artificial  culture, 
provided  that  the  tubes  be  kept  in  the  dark  and  be  prevented  from  drying 
up.  Under  these  conditions  agar  cultures  will  be  found  to  be  living  after 
5  months. 

The  cholera  vibrio  is  very  rapidly  killed  by  drying,  especially  in  artificial 
cultures. 

A  temperature  of  50°-60°  C.  kills  the  vibrios  in  ten  minutes  :  on  the  other 
hand  very  low  temperatures  ( -  10°  C.)  have  no  action  on  their  vitality. 

The  vibrio  is  very  susceptible  to  the  action  of  acids  and  antiseptics  : 
traces  of  perchloride  of  mercury,  quinine  sulphate,  etc.,  arrest  growth  in 
culture. 

Cholera  vibrios  have  been  found  to  be  capable  of  living  for  from  15-30 
days  in  spring  water  (Strauss  and  Dubarry).  They  are  destroyed  in  three  or 
four  days  in  excreta  by  the  action  of  putrefactive  bacteria  (Koch). 

In  discussing  the  disease  experimentally  produced  in  animals  stress  has 


494  THE   CHOLERA  VIBRIO 

already  been  laid  on  the  variations  in  the  virulence  of  the  vibrios.  The 
virulence  of  the  organism  rapidly  becomes  attenuated  in  cultures  but  can  be 
increased  by  passage  through  susceptible  animals  (vide  infra  Toxin). 

Cholera  vibrios  are  very  susceptible  to  the  action  of  micro-organisms  with 
which  they  may  be  associated  (Metchnikoii).  It  has  already  been  pointed 
out  that  certain  organisms  favour  the  growth  in  culture  of  the  cholera  vibrio  ; 
there  are  others  however  which  have  an  adverse  influence,  e.g.  Bacillus 
pyocyaneus,  and  a  white  coccus  isolated  from  water.  This  white  coccus  has 
a  remarkable  effect  on  the  vibrio  ;  during  the  first  few  days  it  prevents  any 
growth  at  all  taking  place  ;  after  a  little  while  the  organism  begins  to  grow, 
but  the  colonies  are  few  in  number  and  small  in  size  and  composed  not  of 
"  comma  "  bacilli  but  of  double  club-shaped  involution  forms  (Metchnikoff). 

2.  Bio-chemical  reactions. 

Nitroso-indol  reaction. — In  cultures  in  peptone  water  the  cholera  vibrio 
reduces  nitrates  to  nitrites  and  produces  indol.  If  a  mineral  acid  free  from 
nitrous  compounds  be  added  to  a  peptone  water  culture  of  the  cholera  vibrio 
a  characteristic  red  reaction  develops,  known  as  the  cholera-red  or  nitroso- 
indol  reaction  (reaction  of  Bujwid  and  of  Salkowski). 

The  reaction  is  most  striking  if  a  little  nitrate  of  potassium  be  added  to  the  peptone 
water.  A  useful  medium  in  which  to  grow  the  vibrio  in  order  to  obtain  the  cholera- 
red  reaction  is  composed  of : 

Peptone  (Chapoteaut  or  Witte),1  10  grams. 

Common  salt,  -  5       ,, . 

Potassium  nitrate,     -  1       „ 

Water      -         -  -      1,000       „ 

This  solution  is  alkaline  in  reaction  and  does  not  require  the  addition  of  soda. 
Sterilize  at  115°  C. 

Sow  the  peptone- water  solution  with  the  vibrio  and  incubate  at  37°  C.  : 
after  24  hours'  incubation  add  gently  1-2  c.c.  of  pure  sulphuric  or  hydro- 
chloric acid.  The  peptone  solution  acquires  a  pink  tint  which  deepens  in 
intensity  during  the  next  few  hours. 

Not  all  cholera  vibrios  give  the  nitroso-indol  reaction,  while  on  the  other 
hand  the  reaction  is  produced  by  certain  other  micro-organisms  which  are  not 
cholera  vibrios. 

Indol  reaction. — The  cholera  vibrio  produces  indol  in  cultures  (p.  374). 

3.  Toxin. 

Cholera  is  an  acute  toxaemia  caused  by  the  absorption  of  a  toxin  elaborated 
by  the  cholera  vibrio  in  the  intestine.  The  toxin  of  cholera  has  for  years 
been  a  subject  of  investigation. 

I.  Brieger  and  Frankel  described  the  occurrence  of  an  albuminoid  substance 
of  unknown  composition — tox-albumin — in  cultures  of  the  cholera  vibrio ; 
this  substance  they  regarded  as  a  diastase.  Utchinsky  showed  that  the 
same  toxin  is  elaborated  in  an  exclusively  mineral  medium. 

Petri  prepared  a  toxin  which  led  to  a  fatal  result  in  guinea-pigs  when 
inoculated  intra-peritoneally  in  doses  of  2  c.c.  The  organism  was  grown  on 
a  5  per  cent,  peptone  solution  and  sterilized  at  120°  C.  ;  it  follows  therefore 
that  the  cholera  toxin,  which  Petri  describes  as  toxo-peptone,  is  not  destroyed 
at  the  temperature  of  boiling  water,  and  for  .this  reason  is  essentially  different 
in  its  nature  from  diphtheria  toxin. 

1  A  good  quality  peptone  must  be  used.  It  is  said  that  the  presence  of  glucose  in 
some  brands  of  peptone  prevents  the  formation  of  indol  and  so  leads  to  a  negative  reaction 
Gorini). 


TOXIN  495 

Hueppe  and  Scholl  grew  the  vibrio  in  the  interior  of  fowls'  eggs  (p.  53,  A.),  the 
object  being  to  cultivate  it  under  conditions  similar  to  those  obtaining  in  the  human 
intestine.  After  incubation  the  contents  of  the  eggs  were  precipitated  with  alcohol 
and  the  precipitate  dissolved  in  sterile  water  ;  this  solution  was  highly  toxic  and 
killed  guinea-pigs  in  a  few  minutes.  Gruber  and  Wiener  however  showed  that  this 
result  was  in  no  way  specific  but  can  be  produced  by  the  sulphuretted  hydrogen 
developed  in  the  cultures  and  by  the  alcohol  used  in  the  precipitation. 

II.  Gamaleia  maintains  that  there  are  several  cholera  toxins.     This  observer 
grew  the  vibrio  in  calf's-foot  broth  for  a  fortnight  at  37°  C.  and  then  left 
the  cultures  standing  for  some  days  at  the  temperature  of  the  laboratory  to 
allow  the  intra-cellular  products  to  diffuse.     This  fluid  is  said  to  contain 
two  toxins  :    one  thermolabile,  producing  diarrhoea  in  rabbits  ;    the  other 
thermostable,  killing  rabbits  without  any  symptoms  of  diarrhoea. 

In  Pfeiffer's  view,  the  toxin  is  contained  within  the  bodies  of  the  vibrios 
themselves  (endotoxin)  and  only  diffuses  on  the  death  and  disintegration  of 
the  organism  :  the  toxins  found  in  solution  in  cultures  are  according  to  this 
observer  merely  a  more  or  less  modified  product  derived  from  the  bodies  of 
the  vibrios. 

Schmitz,  Jurro,  Blell,  extracted  a  nucleo-protein  from  cholera  vibrios 
which  was  very  toxic  to  guinea-pigs  (p.  497).  The  procedure  was  similar  to 
that  adopted  by  Lustig  and  Galeotti  in  the  case  of  plague  (p.  470).  The 
vibrios  were  dissolved  in  a  1  per  cent,  solution  of  potash  and  the  solution 
precipitated  with  acetic  acid. 

Krawkoff  by  a  different  method  extracted  a  similar  nucleo-protein  from 
unfiltered  broth  cultures. 

III.  Metchnikoff,  Roux,  and  Taurelli-Salimbeni  prepared  a  very  powerful 
soluble   toxin   which   diffuses   during   the   life   of  the   organisms:     Ransom 
obtained  similar  results. 

Toxin  of  Roux,  Metchnikoff  and  Taurelli-Salimbeni. 

Preparation  of  the  toxin. — 1.  Select  a  highly  toxigenic  vibrio. 

It  is  best  to  isolate  a  vibrio  direct  from  a  patient  suffering  from  cholera  and  one 
which  has  not  been  passed  through  animals.  Vibrios  isolated  from  man  are  generally 
very  toxigenic,  and  to  retain  their  virulence  they  should  be  sub-cultivated  at  room 
temperature  on  agar  but  as  seldom  as  possible. 

Vibrios  which  have  been  passed  through  the  peritoneal  cavities  of  guinea-pigs  to 
increase  their  virulence  are  generally  only  feebly  toxigenic,  but  their  toxin-producing 
capacities  can  be  augmented  by  passages  in  collodion  sac  cultures  in  guinea-pigs. 
After  filling  a  collodion  sac  with  peptone  water  sow  it  with  the  vibrio  (p.  175)  and  then 
introduce  it  with  aseptic  precautions  into  the  peritoneum  of  a  guinea-pig  ;  leave 
it  for  48  hours  and  then  inoculate  the  contents  directly  into  the  peritoneal  cavity 
of  another  guinea-pig  :  sow  a  second  sac  with  the  peritoneal  exudate  from  this 
second  animal  and  repeat  the  operation  several  times,  each  intra- peritoneal 
incubation  in  collodion  sacs  being  alternated  with  direct  inoculation  into  the  peri- 
toneal cavity.  In  this  way  the  vibrio  acquires  such  a  degree  of  virulence  that  the 
contents  of  the  collodion  sac  rapidly  kill  a  medium-sized  guinea-pig  when  inoculated 
intra- peritoneally  in  doses  of  0'006  c.c. 

The  virulence  is  maintained  by  making  collodion  sac  cultures  in  the  peritoneum 
every  third  or  fourth  day. 

2.  Sow  the  organism  in  the  following  medium  : 

Martin's  peptone  solution,  -       1000  grams. 

Gelatin,  -  20       „ 

Boil  the  solution  to  dissolve  the  gelatin,  make  absolutely  neutral  to  litmus  and 
add  12  c.c.  of  normal  soda  solution.  Heat  at  115°  C.  for  20  minutes,  filter  through 
Chardin  paper,  distribute  in  flasks  and  sterilize  at  110°-112°C.  for  20  minutes. 

When  cool,  add  to  the  contents  of  each  flask  one-fourth  its  volume  of  horse  serum. 
The  blood  should  have  been  collected  3  weeks  previously  and  the  serum  should  be 


496  THE   CHOLERA   VIBRIO 

left  standing  on  the  clot  in  the  ice  chest  for  a  week  before  being  decanted.  In  the 
original  method  there  was  no  serum  in  the  culture  medium,  its  addition  being  sug- 
gested by  the  investigations  of  Brau  and  Denier  (vide  infra). 

Distribute  the  medium  in  Roux  bottles — 50  c.c.  in  each — and  heat  the  latter 
with  their  contents  at  60°  C.  for  3  hours  to  destroy  the  bactericidal  substances  in  the 
serum. 

Sow  the  medium  liberally  with  16  or  18-hour  agar  cultures  and  incubate 
at  38°  C.  Growth  takes  place  in  the  form  of  a  thin  pellicle  and  the  contents 
should  be  shaken  daily  for  the  first  4  days  to  increase  the  aeration.  Filter 
the  culture  on  the  seventh  day  (when  the  content  of  toxin  is  at  its  maximum) 
first  through  paper  then  through  a  Chamberland  filter. 

Brau  and  Denier  state  that  a  very  constant  yield  of  toxin  can  be  obtained  in  the 
following  medium : 

Sterile  Martin's  gelatin-broth,     -  45  c.c. 

Normal  horse-serum  (3  weeks  old),      -  45     „ 

Defibrinated  horse-blood  (3  weeks  old),        -  10     „ 

Mix  and  heat  the  mixture  at  60°  C.  for  3  hours  and  distribute  in  Roux  bottles.  Sow 
the  medium  freely  and  filter  after  incubating  for  7  days,  at  39°  C. 

Properties  of  the  toxin. — The  filtrate  obtained  is  alkaline  and  has  a  charac- 
teristic smell :  it  kills  guinea-pigs  in  doses  of  Ol-0'3  c.c.  per  100  grams  of 
body  weight.  The  toxin  is  unchanged  by  heating  at  100°  C.  for  20  minutes. 
Its  toxicity  is  diminished  by  exposure  to  air — especially  in  presence  of  sun- 
light— but  it  retains  its  properties  if  stored  in  tubes  which  are  exactly  filled, 
sealed  in  the  blow-pipe  flame  and  kept  in  the  dark.  Absolute  alcohol  and 
ammonium  sulphate  precipitate  the  active  principle  from  the  solution. 

Action  of  the  toxin  on  animals. — Small  or  medium-sized  guinea-pigs  are 
more  susceptible  to  cholera  toxin  than  any  other  animal :  large  guinea-pigs 
are  more  immune.  The  toxin  is  equally  virulent  whether  inoculated  sub- 
cutaneously  or  intra-peritoneally  ;  death  takes  place  in  10-30  hours  after 
inoculation  of  medium-sized  doses  (1  c.c.  sub-cutaneously  or  0*3  c.c.  intra- 
peritoneally  for  guinea-pigs  weighing  250  grams).  By  using  a  large  quantity 
of  toxin  a  fatal  result  can  be  obtained  in  a  few  minutes,  especially  if  it  be 
inoculated  intra-peritoneally. 

The  symptoms  are  similar  to  those  which  follow  the  inoculation  of  living  cultures 
of  the  organism,  but  the  incubation  period  is  shorter  :  immediately  after  inoculation 
the  temperature  falls  to  below  normal  and  continues  low  until  death  occurs,  when  it 
may  have  fallen  to  24°  or  25°  C.  Post  mortem,  there  is  a  little  oedema  at  the  site  of 
inoculation,  a  little  fluid  in  the  peritoneum,  hyperaemia  of  the  small  intestine  and 
stomach  and  congestion  of  the  abdominal  viscera :  the  intestine  is  distended  with 
liquid  diarrhceal  matter.  When  the  quantity  of  toxin  inoculated  is  very  small 
there  is  a  temporary  rise  of  temperature  followed  by  a  fall  to  below  normal  and  the 
animal  recovers. 

Rabbits  are  weight  for  weight  more  resistant  than  guinea-pigs  :  for  a 
rabbit  the  dose  must  be  one-third  greater  than  that  required  to  kill  a  similar 
weight  of  guinea-pig.  A  fatal  result  in  adult  rabbits  can  only  be  produced  by 
in tra- venous  inoculation. 

Mice,  rats,  pigeons  and  fowls  are  almost  immune. 

4.  Vaccination. 

The  ease  with  which  laboratory  animals  can  be  vaccinated  against  the 
cholera  vibrio  has  for  a  long  time  stimulated  attempts  to  vaccinate  man. 
Numerous  methods  have  been  described  but  only  two,  Ferran's  and  Haff- 
kine's,  have  been  applied  on  a  large  scale,  and  the  evidence  adduced  as  to 
the  value  of  these  is  based  entirely  upon  statistics. 

I.  Ferran's  vaccine. — Ferran  demonstrated  that  guinea-pigs  which  have 


VACCINATION  497 

survived  the  sub-cutaneous  inoculation  of  a  small  dose  of  a  cholera  culture 
are  immune  to  fatal  doses.  He  therefore  applied  this  method  of  immuniza- 
tion to  man  and  has  performed  more  than  50,000  vaccinations.  The  results 
show  that  after  two  or  three  sub-cutaneous  inoculations  of  his  cultures  (which 
give  rise  to  a  slight  febrile  reaction)  the  vaccinated  individuals  are  immune 
against  cholera. 

For  the  preparation  of  the  vaccine  Ferran  uses  a  vibrio  isolated  from 
cholera  stools  by  the  gelatin  plate  method.  The  vibrio  is  grown  in  broth 
at  37°  C.  and  for  vaccination  purposes  quantities  of  1,  1-5  and  2  c.c.  of  the 
culture  are  inoculated  successively  at  intervals  of  5  days. 

The  results  of  Ferran's  prophylactic  vaccination  have  been  much  criticized. 
At  all  events  he  was  the  first  to  attempt  the  vaccination  of  man  against 
cholera,  and  the  methods  advocated  since  are  nothing  more  than  modifications 
of  his  procedure. 

Gamaleia  suggested  the  use  of  cultures  killed  by  heat  in  place  of  living 
organisms.  TamanchetT  employed  cultures  killed  by  carbolic  acid. 

II.  Haffkine's   vaccine. — Haffkine   considers   that   to   ensure   satisfactory 
vaccination  against  cholera  a  fixed  virus  of  increased  virulence  should  be 
used. 

To  immunize  guinea-pigs  Haffkine  inoculates  them  first  with  an  attenuated 
strain  and  afterwards  with  a  strain  which  has  been  increased  in  virulence. 
He  takes  a  vibrio  whose  virulence  has  been  raised  and  fixed  by  twenty  passages 
through  the  peritoneal  cavities  of  guinea-pigs  and  attenuates  it  by  sub- 
cultivating  it  several  times  in  broth  at  39°  C.  in  a  current  of  air.  The  cultures 
thus  obtained  produce  only  a  local  and  general  reaction  on  sub-cutaneous 
inoculation  into  the  guinea-pig.  The  animal  is  then  inoculated  sub-cutaneously 
with  the  virus  of  increased  virulence,  which  is  found  to  cause  no  disturbance 
of  its  health.  After  this  it  is  immune  against  all  methods  of  infection  with 
the  vibrio  (sub-cutaneous  and  intra-peritoneal  inoculation  and  ingestion). 

This  method  nevertheless  failed  to  immunize  ground-squirrels  and  suckling 
rabbits  against  experimental  intestinal  cholera  (Zabolotny,  MetchnikofE). 

To  vaccinate  man  against  intestinal  cholera,  Haffkine  proposed  inoculating 
beneath  the  skin  first  an  attenuated  virus  and  a  week  later  one  of  his  virulent 
cultures  (^th.  to  TV*n  °f  an  &gar  culture).  This  method  was  subsequently 
modified  and  he  now  gives  a  single  inoculation  of  a  virulent  virus  recently 
recovered  from  the  peritoneum  of  a  guinea-pig.  In  the  case  of  an  adult  he 
injects  0*5  c.c.  of  an  emulsion  of  an  agar  culture  in  about  5  c.c.  of  sterile 
water.  The  results  obtained  by  Haffkine,  Powell,  Simpson,  Wright  and  others 
seem  very  satisfactory. 

III.  Kolle's  vaccine. — For  human  vaccination,  Kolle  uses  agar  cultures 
made  into  an  emulsion  (with  sterile  normal  saline  solution)  and  heated  at 
56°  C.  for  an  hour.    The  inoculation  of  T\yth  to  ith  of  an  agar  culture  sets  up  a 
painful  inflammation  at  the  site  of  inoculation  which  lasts  2  or  3  days.     From 
the  fifth  day  after  inoculation  the  serum  of  the  inoculated  individual  is  endowed 
with  both  bactericidal  and  bacteriolytic  properties,  and  these  can  still  be 
demonstrated  so  long  as  a  year  afterwards. 

According  to  Kolle  it  is  a  matter  of  indifference  in  the  case  of  the  guinea-pig 
whether  a  virulent  or  non- virulent  vibrio  be  used.  This  fact  confirms  Ferran, 
who  considers  that  no  value  attaches  to  increasing  the  virulence  of  the  virus 
by  animal  passage  for  the  preparation  of  human  vaccines. 

IV.  Vaccination  with  bacterial  extracts. — The  extracts  prepared  by  Schmitz, 
Turro,  Blell  (p.  495)  are  toxic  for  guinea-pigs :  10-15  mg.  is  a  fatal  dose,  but 
if  smaller  doses  (1-5  mg.)  be  used  the  animal  is  immune  for  several  months 
against  both  the  toxin  and  the  organism.     For  human  vaccination  2  mg. 

2i 


498  THE  CHOLERA  VIBRIO 

of  the  nucleo-protein  dissolved  in  1  c.c.  of  slightly  alkaline  water  are  inoculated 
sub-cutaneously. 

Strong's  vaccine  for  human  inoculation  is  prepared  by  making  an  emulsion 
in  sterile  water  of  vibrios  from  an  agar  culture,  sterilizing  at  60°  C.,  macerating 
at  37°  C.  for  2  days,  and  filtering  through  a  Reichel  bougie.  In  large  doses 
the  product  is  fatal  to  rabbits  but  in  smaller  doses  acts  as  a  vaccine.  If 
inoculated  in  doses  of  3-4  c.c.  in  man  it  produces  a  slight  reaction  and  the 
serum  of  the  inoculated  person  is  afterwards  bactericidal  and  agglutinating. 

V.  Besredka's  vaccine. — Besredka  succeeded  in  conferring  rapidly  a  lasting 
immunity  on  rabbits  without  setting  up  an  inflammatory  reaction  by  inocu- 
lating them  sub-cutaneously  with  a  vaccine  prepared  by  sensitizing  the  bodies 
of  the  vibrios  with  an  anti-choleraic  serum  (see  typhoid  bacillus  and  plague 
bacillus). 

VI.  Vaccination  against  choleraic  peritonitis. — Guinea-pigs  are  very  easily 
immunized    against    peritoneal    infection.     Intra-peritoneal    inoculation    of 
vibrios  killed  by  heat  is  efficient  in  preventing  a  subsequent  choleraic  peri- 
tonitis.    To  obtain  a  lasting  immunity  the  single  inoculation  of  dead  vibrios 
should  be  supplemented  by  several  inoculations  of  living  organisms. 

Klein  has  shown  that  the  products  of  organisms  other  than  the  cholera  vibrio  can 
induce  immunity  to  choleraic  peritonitis :  intra-peritoneal  inoculation  of  small 
doses  of  a  heated  culture  of  the  Micrococcus  prodigiosus  will  immunize  guinea-pigs 
against  fatal  doses  of  the  vibrio.  Israel  has  found  that  sterile  broth  inoculated 
intra-peritoneally  will  also  immunize  guinea-pigs  against  choleraic  peritonitis. 
Inoculations  of  human  serum  (vide  infra),  of  normal  saline  solution  or  of  urine  all 
act  in  a  similar  manner  (Israel,  Metchnikoff).  The  explanation  of  these  phenomena 
is  to  be  found  in  the  fact  that  the  substances  inoculated  stimulate  phagocytosis,  with 
the  result  that  the  leucocytes  ingest  the  vibrios  and  so  arrest  the  peritoneal  infection. 

VII.  Vaccination  with  toxin. — Metchnikoff,  Roux  and  Taurelli-Salimbeni 
showed  that  animals  very  quickly  become  accustomed  to  cholera  toxin  and 
that  guinea-pigs,  rabbits,  horses  and  goats  may  be  immunized  by  inoculating 
them  with  toxin. 

When  guinea-pigs  and  rabbits  are  treated  with  small  doses  of  toxin  the  temperature 
rises  for  a  short  period  after  each  inoculation  and  then  falls  below  normal  for  about 
20  hours.  Repeated  inoculations  are  accompanied  by  a  certain  loss  of  weight  from 
which  the  animals  soon  recover,  though  rabbits  are  slower  in  picking  up  than  guinea- 
pigs  :  the  inoculations  should  be  withheld  until  the  animals  have  regained  their 
original  weights.  It  is  somewhat  difficult  to  immunize  rabbits  satisfactorily. 

The  inoculation  of  2-4  c.c.  into  goats  is  followed  by  a  rise  of  temperature  which 
becomes  less  marked  as  the  animal  becomes  accustomed  to  the  inoculations. 

Horses  react  sharply  to  sub-cutaneous  inoculation  of  the  toxin  and  show  a  well- 
marked  and  persistent  oedema  at  the  site  of  inoculation.  For  purposes  of  immuniza- 
tion it  is  better  to  adopt  the  intra-venous  method,  commencing  with  very  small 
doses  (always  dilute  the  toxin  with  an  equal  volume  of  normal  saline  solution  on 
account  of  the  high  degree  of  alkalinity  of  the  former).  Inoculation  into  the  veins 
is  followed  by  a  more  or  less  marked  reaction  (fever,  diarrhoea,  etc.)  ;  an  interval 
of  about  10  days — sufficient  to  allow  the  animal  to  recover  completely  from  the  pre- 
vious dose — should  elapse  between  each  two  inoculations.  After  about  6  months, 
50-60  c.c.  can  be  given  at  a  single  inoculation.  The  horse  is  bled  12  days  after  the 
last  inoculation. 

5.  Serum  therapy. 

The  serum  of  animals  vaccinated  against  the  cholera  vibrio  exhibits  pro- 
phylactic properties  (Klemperer). 

Lazarus  has  shown  that  the  serum  of  persons  who  have  recovered  from  an 
attack  of  cholera  also  possesses  considerable  prophylactic  properties  :  in 
some  cases  1  c.c.  of  serum  will  protect  a  guinea-pig  against  choleraic  peri- 
tonitis. The  prophylactic  property  of  the  blood  however  plays  no  part  in 


SERUM  THERAPY  499 

the  cure  of  human  cholera,  for  Metchnikoff  and  Klemperer  have  shown  that 
the  serum  of  persons  who  have  never  suffered  from  cholera  is  sometimes 
prophylactic,  that  the  prophylactic  property  of  the  blood  may  be  very  highly 
developed  in  the  blood  of  persons  who  have  just  died  of  cholera,  and  that  it 
is  on  the  other  hand  often  absent  in  persons  convalescent  from  the  disease 
(Metchnikoff). 

I.  Antibacterial    serum. — Pfeiffer    vaccinated    guinea-pigs    with    cholera 
vibrios  (p.  498)  and  obtained  a  serum  active  in  -^  mg.— that  is  to  say  a  serum 
of  which  T\  mg.  was  sufficient  to  vaccinate  a  guinea-pig  against  choleraic 
peritonitis  if  injected  either  before,   or   at   the    same  time  as,   or    within 
5  minutes  of  the  vibrios.     The  serum  is  bactericidal  and  agglutinates  the 
cholera  vibrio  (vide  infra). 

Metchnikoff  has  shown  that  Pfeiffer's  serum  has  no  antitoxic  properties  : 
it  is  very  efficient  in  protecting  the  blood  and  organs  against  infection  because 
it  stimulates  phagocytosis  and  allows  the  leucocytes  to  ingest  the  organisms, 
but  it  is  absolutely  useless  against  intestinal  cholera  which  is  an  intoxication. 

Reaction  of  immunity. — Pfeiffer  has  suggested  using  the  immunizing  pro- 
perty of  the  serum  of  vaccinated  animals  as  a  means  of  differentiating  the 
cholera  vibrio  from  allied  species  :  according  to  Pfeiffer,  the  serum  of  an 
animal  vaccinated  against  cholera  protects  guinea-pigs  against  infection  with 
the  cholera  vibrio  but  not  against  closely  related  organisms.  To  determine 
the  nature  of  a  vibrio  then  it  is  sufficient  to  inoculate  the  organism  under 
investigation  into  a  guinea-pig  treated  with  anticholera  serum  :  it  is  said 
that  the  animal  will  only  resist  infection  when  the  vibrio  inoculated  belongs 
to  the  choleragenic  group.1  Metchnikoff  has  shown  that  this  test  is  of  but 
little  value  :  the  reaction  of  immunity  may  fail  in  the  case  of  vibrios  isolated 
from  the  stools  of  cholera  patients  and  be  present  in  the  case  of  saprophytic 
vibrios. 

II.  Metchnikoff,   Roux   and   Taurelli-Salimbeni's   serum. — The   serum   of 
horses  immunized  with  toxin  according  to  the  directions  of  these  observers 
(vide  ante}  is  bactericidal  and  agglutinating  :    it  is  also  prophylactic  against 
choleraic  peritonitis  in  guinea-pigs,  the  prophylactic  dose  lying  between  0*01 
and  O'OOS  c.c.     It  is  antitoxic  and  protects  against  intestinal  cholera. 

This  serum,  if  inoculated  sub-cutaneously  into  small  rabbits  in  doses  of 
4—8  c.c.  before  feeding  them  with  cholera  vibrios,  will  protect  them  against 
intestinal  cholera.  Out  of  100  rabbits  treated  with  the  serum  56  escaped 
infection  while  only  16  per  cent,  of  the  controls  survived.  The  serum  is 
efficient  if  administered  at  the  same  time  as  the  animals  are  fed  with  the 
virus  but  it  has  no  effect  at  all  if  the  animals  be  fed  24  hours  before  the 
administration  of  the  serum.  The  value  of  this  serum  in  human  cholera 
has  yet  to  be  proved. 

6.  Bactericidal  properties.    Agglutination. 

1.  Pfeiffer  was  the  first  to  demonstrate  the  bactericidal  and  agglutinating 
properties  of  the  serum  of  immunized  animals  in  vivo. 

Pfeiffer's  phenomenon. — If  an  emulsion  of  cholera  vibrios  be  inoculated 
into  the  peritoneal  cavity  of  a  guinea-pig  immunized  against  the  vibrio,  the 
peritoneal  fluid  will  in  a  short  time — 10-30  minutes — be  found  on  microscopical 
examination  to  contain  only  non-motile  small  more  or  less  granular  spherical 
micro-organisms. 

Experiment. — Inject  into  the  peritoneal  cavity  of  an  immunized  guinea-pig  2  c.c. 
of  sterile  broth  in  which  one- half  of  an  agar  culture  of  the  vibrio  to  be  examined 

1  A  control  guinea-pig  which  has  not  been  treated  with  the  cholera  serum  must  of 
course  be  inoculated  at  the  same  time  with  the  vibrio  under  investigation. 


500  THE   CHOLERA   VIBRIO 

has  been  emulsified.  Ten  minutes  afterwards  withdraw,  with  a  very  fine-pointed 
pipette,  a  few  drops  of  the  peritoneal  fluid  and  examine  it  under  the  microscope 
(either  stained  or  unstained).  If  the  vibrios  are  motile  and  have  retained  their 
characteristic  shape,  the  reaction  is  negative,  and  the  vibrio  is  not  a  cholera  vibrio. 
If  on  the  other  hand  only  non-motile  spherical  dots  are  seen,  then  the  vibrio  may 
be  regarded  as  a  true  cholera  vibrio  (vide  also  p.  227). 

If  there  be  no  immunized  guinea-pig  at  hand,  inoculate  a  normal  guinea- 
pig  intra-peritoneally  with  an  emulsion  of  the  vibrio  mixed  with  a  little  tested 
anticholera  serum.  The  same  granular  change  will  be  seen  as  in  the  fore- 
going case,  and  in  addition  the  organisms  will  be  agglutinated  into  small 
clumps. 

2.  The  vibrios  are  equally  agglutinated  and  disintegrated  by  an  anti- 
cholera  serum  in  vitro  (Metchnikoff,  Bordet). 

To  demonstrate  this  phenomenon  rub  up  a  small  quantity  of  an  agar  culture  in  a 
little  sterile  broth  and  examine  under  the  microscope  to  make  sure  there  are  no 
clumps :  then  add  5  to  10  per  cent,  of  the  serum  and  in  a  few  minutes  agglutina- 
tion followed  by  granulation  of  the  bacilli  will  occur,  the  result  being  most  marked 
when  the  mixture  has  been  incubated  for  2  hours  at  37°  C. 

An  agglutinating  serum  can  be  easily  obtained  by  inoculating  cultures 
killed  by  heat  into  the  peritoneal  cavity  or  into  the  veins  of  a  guinea-pig 
or  rabbit. 

Applications. — The  phenomenon  of  agglutination  furnishes  a  means  of 
identifying  the  vibrio  :  as  a  rule,  Pfeiffer's  phenomenon  on  the  one  hand  and 
agglutination  in  vitro  on  the  other  occur  only  with  cholera  vibrios  and  not 
with  closely  related  species.  In  practice  it  will  be  sufficient  to  perform  one 
of  these  tests  because  no  vibrio  has  yet  been  encountered  which  gives  one  and 
not  the  other  and  vice  versa.  Unfortunately  the  value  of  the  method  is  not 
absolute  since  it  sometimes  fails  with  vibrios  isolated  from  the  stools  of  cholera 
patients  and  may  be  produced  with  vibrios  devoid  of  all  pathogenic  properties. 

Serum  diagnosis. — The  serum  of  persons  suffering  from  cholera  will  agglu- 
tinate an  emulsion  of  the  cholera  vibrio  in  5-60  minutes  in  a  dilution  of  1  in 
20  (Achard  and  Bensaude).  This  reaction  furnishes  a  rapid  method  of 
diagnosis,  but  it  has  to  be  remembered  that  normal  human  serum  may 
agglutinate  the  vibrio  in  a  dilution  of  1  in  10  (Pfeiffer  and  Kolle). 

7.  Complement  fixation. 

Cholera  serum  contains  immune  bodies  (sensibilisatnces]  specific  for  the 
vibrio.  The  complement  fixation  reaction  applied  according  to  the  method 
of  Bordet  and  Gengou  (p.  232)  gives  very  accurate  results  in  differentiating 
the  cholera  vibrio  from  organisms  allied  to  it  (de  Besche  and  Kon). 

SECTION  IV.- DETECTION,   ISOLATION  AND  IDENTIFICATION 
OF  THE   CHOLERA  VIBRIO. 

The  detection  and  isolation  of  vibrios  in  general  is  easy,  but  identification 
of  the  cholera  vibrio  in  particular  presents  considerable  difficulties. 

1.  Detection  and  isolation. 
A.  Microscopical  examination. 

This  method  of  investigation  is  applicable  only  to  the  examination  of  stools, 
or  of  the  exudates  and  tissues  of  animals. 

Detection  in  the  excreta. — Take  one  of  the  small  mucous  or  riziform  particles, 
spread  it  on  a  slide  and  stain  with  dilute  carbol-fuchsin.  In  typical  cases 
this  is  a  conclusive  test — the  vibrios  are  found  in  almost  pure  culture  arranged 


ISOLATION   OF  THE   VIBRIO  501 

in  swarms  all  pointing  the  same  way  like  fish  in  water  ;  but  frequently  micro- 
scopical examination  furnishes  no  certain  evidence.  Microscopical  examina- 
tion should  always  be  supplemented  by  sowing  cultures  with  traces  of  the 
stools. 

Very  pretty  microscopical  preparations  can  be  made  by  double  staining  the  film 
by  Gram's  method  using  dilute  carbol-fuchsin  as  a  counterstain :  the  cholera  vibrio 
is  red,  and  the  gram-positive  organisms  violet. 

Dunbar's  method. — Dunbar  applies  the  phenomenon  of  agglutination  to  the 
detection  of  the  vibrios  in  the  stools.  Prepare  two  hanging- drop  cultures  with  a 
trace  of  the  suspected  material  diluted  in  a  drop  of  peptone  water,  and  add  to  one 
a  drop  of  an  agglutinating  serum.  On  microscopical  examination,  the  vibrios  are 
motile  in  the  preparation  to  which  no  serum  was  added  and  non-motile  and  agglu- 
tinated in  the  other.  Whether  the  method  be  reliable  or  no,  the  technique  certainly 
requires  much  care. 

Staining  of  sections.— Sections  of  the  intestine  are  best  stained  by  Nicolle's 
tannin  method. 

B.  Isolation. 

Stools. — The  method  originally  employed  consisted  in  isolating  the  organism 
from  the  stools  by  means  of  gelatin  or  agar  plates  ;  but  the  plates  are  very 
often  overgrown  with  other  organisms  so  that  the  vibrio  cannot  be  isolated. 
The  following  method  is  preferable. 

Metchnikoff 's  method. — 1.  Prepare  a  number  of  tubes  of  gelatin-peptone-salt 
solution  (p.  33),  and  after  sowing  them  with  a  trace  of  the  suspected  stool 
incubate  them  at  37°  C. 

2.  After  3  or  4  hours  the  tubes  will  be  cloudy  and  in  about  7  hours  a  thin 
pellicle  will  have  formed  on  the  surface  of  the  medium.     Examine  a  trace 
of  the  film  under  the  microscope  :    as  a  rule  the  vibrio  will  not  be  found  in 
pure  culture  but  mixed  with  various  other  organisms. 

3.  To  obtain  a  pure  culture  sub-cultivate  a  trace  of  the  pellicle  in  another 
tube  of  the  gelatin -peptone-salt  solution,  incubate  for  6  or  7  hours  and  if 
necessary  (after  microscopical  examination  of  a  film  from  the  pellicle)  sow 
another  tube  in  the  same  way  ;    it  is  however  usually  sufficient  to  plate  on 
agar  after  the  first  sub-cultivation. 

Liquefy  a  tube  of  agar  and  pour  the  medium  into  a  Petri  dish,  allow  it  to 
set  and  then  sow  surface  stroke  cultures  with  a  trace  of  the  material  from  the 
pellicle.  Incubate  at  37°  C.  After  a  few  hours,  small  delicate  transparent 
or  opalescent  but  never  opaque  colonies  are  visible  which  can  'be  picked  off 
after  about  8  hours'  incubation.  The  whole  process  of  isolation  thus  takes 
only  about  15  hours.  / 

Gelatin  plates  may  also  be  prepared  at  the  same  time  as  the  agar  plates 
and  an  examination  of  them  subsequently  will  furnish  a  clue  to  identification. 

[Note. — Instead  of  the  peptone-gelatin  medium,  ordinary  peptone  water  is 
very  commonly  used  for  the  isolation  of  the  cholera  vibrio  and  Ottolenghi  has 
recently  suggested  bile  as  an  "  enrichment  "  medium  for  the  same  purpose.] 

Water.  (Metchnikoff's  method.)  Recommended. — 1.  Prepare  a  series  of 
conical  flasks  of  250  c.c.  capacity  or  thereabouts.  Measure  200  c.c.  of  water 
into  each  and  make  a  mark  on  the  glass  with  a  diamond. 

2.  To  each  flask  add  the  following  solution  : 


Water,     - 

Peptone  (Chapoteaut), 

Common  salt,  - 

Gelatin,  - 

Solution  of  soda, 


50  c.c. 
2  grams. 
2 

4       „ 
Q.S.  to  make 


faintly  alkaline. 
Sterilize  in  the  autoclave. 


502 


THE   CHOLERA  VIBRIO 


3.  To  the  contents  of  each  flask  add  150  c.c.  of  the  water  under  examina- 
tion (that  is,  up  to  the  mark  on  the  flask)  and  incubate  at  37°  C.  If  the 
water  should  contain  vibrios  a  thin  pellicle  will  form  on  the  surface  of  the 
liquid  in  about  &-10  hours.  A  trace  of  this  film  may  be  examined  micro- 
scopically and  one  or  two  sub-cultures  in  series  can  be  made  in  the  gelatin- 
peptone-salt  medium  [or  in  peptone-water] ;  and  if  need  be  the  organism  can 
be  isolated  on  agar  in  the  manner  described  above. 

2.  Identification. 

Having  isolated  a  vibrio  from  the  excreta  or  from  water  it  is  then  necessary 
to  identify  the  organism,  because  there  are  a  number  of  vibrios  which  must 
be  regarded  as  harmless  saprophytes.  Numerous  species  of  vibrios  have 
been  found  in  different  waters  by  Metchnikoff,  Blachstein,  Sanarelli, — the 
last-named  observer  has  described  no  less  than  32  species  in  the  Paris  water. 

There  is  at  present  no  known  characteristic  which  is  pathognomonic  of  the 
cholera  vibrio,  and  when  vibrios  are  found  in  water  in  the  absence  of  any 
epidemic  of  cholera  it  may  be  impossible  to  identify  them.  The  following 
are  generally  regarded  as  the  classical  characteristics  of  the  cholera  vibrio  : — 

1.  The  appearances  presented  in  gelatin  stab  and  plate  cultures,  p.  493. 

2.  The  presence  and  the  number  of  flagella,  p.  492. 

3.  The  nitroso-indol  reaction  in  peptone-water  cultures,  p.  494. 

4.  The  virulence  for  guinea-pigs  (choleraic  peritonitis),  p.  489. 

5.  The  immunity  reaction,  p.  499. 

6.  Agglutination  by  an  anticholera  serum  and  the  occurrence  of  Pfeifler's 

phenomenon,  p.  500. 

Attention  has  already  been  drawn  in  the  course  of  this  chapter  to  the  lack 
of  specificity  and  constancy  of  these  characters  ;  the  most  reliable  is  the 
agglutination  reaction  with  the  specific  serum.  A  better 
means  of  identifying  the  cholera  vibrio  will  perhaps  consist 
in  feeding  young  rabbits  on  the  suspected  vibrio  either  alone 
or  mixed  with  ancillary  micro-organisms.  It  seems  probable 
that  the  application  of  the  "fixation  of  the  complement" 
reaction  may  be  of  considerable  use  in  the  identification  of 
vibrios. 

The  Vibrio  of  Finkler-Prior. 

This  vibrio  was  discovered  by  Finkler  and  Prior  in  the  stools 
of  a  man  suffering  from  acute  enteritis.  It  was  found  again  by 
the  same  observers  in  the  stools  of  persons  infected  with  cholera 
nostras,  and  it  is  possible  that  this  was  the  organism  found  by 
Ruete  and  Enoch  in  the  stools  of  a  woman  suffering  from  a  fatal 
attack  of  diarrhoea. 

The  vibrio  which  Rommelaer  took  to  be  the  Finkler-  Prior  vibrio 
really  belongs  to  the  group  of  the  true  cholera  vibrios. 

Experimental  inoculation. — When  inoculated  intra-peritoneally 
into  a  guinea-pig  the  Finkler-Prior  vibrio  sets  up  a  fatal  peri- 
tonitis. 

A  fatal  infection  can  be  produced  in  pigeons  by  inoculating 
the  organism  into  the  pectoral  muscle.     In  man  the  consumption 
of  an  agar  culture  of  the  vibrio  after  the  contents  of  the  stomach 
have  been  made  alkaline  produces  slight  intestinal  disturbance 
FIG    242— Vibrio    (Metchnikoff). 
of  Finkler-Prior.       Microscopical   appearance. — Microscopically  the   Tinkler-Prior 
Stab  culture  in  gela-   vibrio  is  similar  to  the  cholera  vibrio  but  the  former  is  slightly 
swollen  in  the  centre  and  tapered  a,t  the  ends.     The  two  organ- 
isms give  the  same  staining  reactions  and  both  are  gram-negative. 
The  Finkler-Prior  vibrio  is  motile  and  has  a  single  flagellum. 


SIMILAR  VIBRIOS  503 

Cultural  characteristics. — Cultures  of  the  Finkler-Prior  vibrio  resemble  those  of 
the  cholera  vibrio.  The  former  liquefies  gelatin  more  rapidly  and  does  not  form 
a  bubble  of  air  on  the  surface  of  the  liquefied  portion ;  it  coagulates  milk. 

Biological  properties. — The  Finkler-Prior  vibrio  produces  indol  but  only  traces 
of  nitrites.  The  nitroso-indol  reaction  is  positive  but  only  slightly  marked  and 
slowly  produced  (several  days). 

The  Vibrio  of  Deneke. 

This  vibrio  was  isolated  by  Deneke  from  an  old  cheese.  Morphologically,  it  is 
similar  to  the  cholera  vibrio  and  in  gelatin  plate  cultures  the  appearance  of  the 
colonies  of  the  two  organisms  may  be  identical.  It  liquefies  gelatin  rather  more 
quickly  than  the  cholera  vibrio  but  less  rapidly  than  the  Finkler-Prior  vibrio. 

Guinea-pigs  die  as  the  result  of  intra-peritoneal  inoculation  of  the  vibrio  (Hueppe, 
Metchnikoff) :  the  organism  is  also  pathogenic  for  pigeons  (Kasanky,  Metchnikoff). 
In  man,  infection  with  the  organism  causes  diarrhoea  (Metchnikoff). 

Deneke's  vibrio  produces  indol  but  very  little  nitrites.  The  cholera-red  reaction 
is  inconstant  and  very  poorly  marked. 

Vihrio  Metchnikowi. 

(Vibrion  avicide.) 

The  Metchnikoff  vibrio  was  discovered  in  Odessa  by  Gamaleia,  in  a  disease  of 
fowls  of  which  it  is  the  cause.  The  disease  is  characterized  by  weakness,  drowsi- 
ness and  diarrhoea  :  post  mortem,  the  alimentary  canal  is  hyperaemic  and  the  small 
intestine  contains  a  yellowish-grey  liquid  which  may  be  blood-stained ;  the  vibrio 
is  found  in  very  large  numbers  in  this  fluid.  As  a  rule,  the  organism  does  not  gain 
access  to  the  blood  stream,  though  in  young  fowls  affected  with  the  disease  it  may 
be  isolated  from  the  blood. 

Experimental  inoculation. — Guinea-pigs  are  more  susceptible  to  the  Metchnikoff 
vibrio  than  to  the  cholera  vibrio  ;  intra-peritoneal  and  sub-cutaneous  inoculation 
and  ingestion — even  without  previous  alkalinization  of  the  gastric  contents — all 
lead  to  a  fatal  result. 

The  Metchnikoff  vibrio  kills  young  fowls  whatever  the  channel  of  infection : 
simple  ingestion  alone  is  fatal.  Adult  birds  are  much  less  susceptible  and  cannot 
be  infected  by  feeding.  Pigeons  though  very  susceptible  to  sub-cutaneous  or  intra- 
muscular inoculation  resist  infection  when  fed  with  the  organism. 

Ingestion  of  the  vibrio  is  harmless  to  man. 

Microscopical  appearance. — Morphologically  the  Metchnikoff  vibrio  is  similar  to 
the  cholera  vibrio :  it  is  motile  and  has  a  single  flageilum ;  sometimes  spirals  of 
4  or  5  turns  are  seen. 

Cultural  characteristics. — The  Metchnikoff  vibrio  grows  on  all  the  ordinary  media 
and  the  growths  are  similar  to  those  of  the  cholera  vibrio.  On  potato,  it  grows 
more  abundantly  than  the  cholera  vibrio  and  forms  a  yellow- brown  streak.  Cul- 
tures in  milk  ultimately  become  very  acid  and  the  casein  is  coagulated  about  the 
eighth  day. 

Biological  properties. — The  Metchnikoff  vibrio  produces  indol  and  nitrites  in 
peptone  solutions  and  gives  a  very  marked  nitroso-indol  reaction.  Guinea-pigs, 
pigeons  and  fowls  can  be  immunized  by  the  inoculation  of  cultures  killed  by  heat 
at  120°  C. 


CHAPTER   XXXIII. 

PFEIFFER'S  INFLUENZA  BACILLUS.  THE  H^MOGLO- 
BINOPHILIC  BACILLI.  THE  BACILLUS  OF 
WHOOPING  COUGH. 

Introduction. 

Section  I.— Experimental  inoculation,  p.  505. 

Section  II. — Morphology  and  cultural  characteristics,  p.  506. 

Section  III. — Biological  properties,  p.  508. 

1.  Vitality  and  virulence,  p.  508.     2.  Toxin,  p.  508.     3.  Immunity  and  serum 

therapy,  p.  508.     4.  Agglutination,  p.  509.     5.  Secondary  infections  and  ancillary 

micro-organisms,   p.  509. 
Section  IV. — Detection  and  isolation  of  the  bacillus,  p.  510. 

The  hsemoglobmophilic  bacilli. 

1.  Pfeiffer's  pseudo-influenza  bacillus  and  similar  organisms,  p.  510.  2.  The 
bacillus  of  acute  contagious  conjunctivitis,  p.  510.  3.  The  bacillus  of  sub-acute 
conjunctivitis,  p.  511. 

The  bacillus  of  whooping  cough,  p.  511. 

THE  Bacillus  influenzce  was  discovered  by  Pfeiffer  whose  investigations 
were  subsequently  confirmed  by  Weichselbaum,  Huber  and  others.1 

Influenza  is  a  disease  peculiar  to  man.  Pfeiffer's  bacillus  is  found  in  the  sputum, 
nasal  mucus  and  respiratory  passages  of  those  suffering  from  the  disease. 

The  organism  may  set  up  foci  of  broncho -pneumonia  in  the  lung  and  may  also 
be  the  cause  of  some  of  the  sequelae  of  the  disease  :  pleurisy,  meningitis,  and  perio- 
stitis (Meunier).  The  bacillus  may  still  be  present  in  the  sputum  some  weeks  after 
all  symptoms  of  the  disease  have  disappeared  and  for  an  even  longer  time  in  chronic 
lesions  of  the  lung — e.g.  tuberculosis  and  other  conditions. 

The  symptoms  point  to  influenza  being  an  intoxication  rather  than  a  generalized 
infection  and  Pfeiffer  was  never  able  to  isolate  the  organism  from  the  blood  stream. 
On  the  other  hand  Meunier  found  Pfeiffer's  bacillus  in  the  blood  in  four  out  of  eight 
cases  of  the  disease  examined  by  him  during  life  and  Ghedini  during  an  epidemic  of 
influenza  isolated  the  organism  from  blood  taken  from  the  bend  of  the  elbow  (64 
out  of  100  cases)  and  from  material  obtained  by  puncture  of  the  spleen  during  the 
febrile  attack  (57  out  of  100  cases).  Post  mortem,  Rosenthal  found  the  bacillus  in 
the  blood  of  the  heart  in  all  the  fatal  cases  he  examined. 

Pfeiffer's  bacillus  has  not  been  found  in  by  any  means  all  cases  of  clinical 
influenza,  many  of  which  are  due  to  infection  with  the  Micrococcus  catarrhalis. 
the  Pneumococcus  and  possibly  with  other  organisms.  Pfeiffer  failed  to  isolate 
the  bacillus  in  the  1899  epidemic,  and  in  a  very  severe  epidemic  at  Eennes 

1  The  micro-organism  found  by  Canon  and  Bruschettini  in  the  blood  of  persons  suffer- 
ing from  influenza  differs  essentially  from  Pfeiffer's  bacillus  in  that  it  is  a  small  strepto- 
coccus which  grows  well  on  ordinary  media  and  is  pathogenic  to  rabbits. 


EXPERIMENTAL  INOCULATION  505 

in  1897-8  Besson  only  found  the  organism  in  80  per  cent,  of  cases  investigated. 
Achalme,  Rosenthal,  Bezan9on,  and  others  have  reported  similar  observations. 
Again,  Pfeiffer's  bacillus  or  an  organism  like  it  has  been  found  in  healthy 
persons  as  well  as  in  cases  of  whooping  cough,  broncho-pneumonia,  etc. 

Relying  on  these  negative  observations  many  authors  have  raised  a  doubt  as  to 
the  specific  relationship  of  Pfeiffer's  bacillus  to  influenza.  Rosenthal,  for  instance, 
comes  to  the  conclusion  that  "  the  haemophilic  cocco-bacillus  (or  bacillus  of  Pfeiffer} 
is  a  micro-organism  commonly  found  among  the  pathological  flora  of  the  lung,  and 
is  not  the  bacillus  which  causes  influenza."  But  the  known  persistence  of  Pfeiffer's 
bacillus  in  chronic  lesions  of  the  lung  offers  quite  a  satisfactory  explanation  of 
the  occurrence  of  the  organism  among  the  flora  of  phthisical  cavities,  tuberculous 
bronchitis,  etc.,  and  is  in  no  way  inconsistent  with  the  specific  relationship  of 
the  bacillus  to  influenza.  Further,  there  is  no  apparent  reason  why,  under  certain 
circumstances,  Pfeiffer's  bacillus  should  not  live  as  a  saprophyte  in  the  human 
tissues  :  it  is  well  known  that  the  pneumococcus,  the  diphtheria  bacillus  and  other 
organisms  are  frequently  found  under  such  conditions,  and  no  question  is  ever  raised 
as  to  the  specific  relationship  of  these  organisms  to  their  respective  diseases.  Again 
new  haemophilic  micro-organisms  similar  to  or  identical  with  Pfeiffer's  bacillus  are 
being  constantly  described,  e.g.  the  organisms  found  by  Jochmann  and  Moltrecht 
in  whooping  cough,  by  Wolff  in  rats,  and  by  Friedberger  in  dogs.  Hence,  it  would 
appear  that  there  is  a  group  of  haemophilic  micro-organisms,  of  which  Pfeiffer's 
bacillus  is  the  type,  which  inhabit  for  preference  the  respiratory  passages  but  exhibit 
very  divergent  pathogenic  properties. 

Ancillary  micro-organisms. — In  influenza,  particularly  in  the  pulmonary 
lesions,  Pfeiffer's  bacillus  is  frequently  accompanied  by  other  pathogenic 
organisms,  the  more  common  being  pneumococci  and  various  streptococci. 
Reference  will  be  made  to  these  associated  micro-organisms  later  but  mean- 
while it  may  be  said  that  they  largely  determine  the  severity  of  the  disease. 


SECTION  I.— EXPERIMENTAL  INOCULATION. 

As  a  result  of  his  observations,  Pfeiffer  came  to  the  conclusion  that  with 
the  exception  of  monkeys  the  lower  animals  are  immune  against  Pfeiffer's 
bacillus.  This  natural  resistance  may,  however,  be  overcome  by  experi- 
mental methods. 

Monkeys. — The  inoculation  of  a  pure  culture  of  Pfeiffer's  bacillus,  or  of 
sputum  from  cases  of  influenza,  into  the  trachea,  lung  or  nasal  fossae  of 
monkeys  is  followed  by  a  disease  with  symptoms  similar  to  those  of  influenza 
in  man.  As  a  rule  the  animal  recovers.  In  one  fatal  experiment,  the  pul- 
monary lesions  were  very  like  those  seen  in  the  human  disease  ;  the  bacillus 
was  found  in  this  case  in  small  numbers  in  the  blood,  bronchial  secretions, 
and  pulmonary  mucus  (Pfeiffer). 

Laboratory  animals. — Rats,  pigs,  cats,  dogs,  and  pigeons  are  absolutely 
immune. 

Rabbits. — The  inoculation  of  large  doses  of  pure  cultures  into  the  ear  vein 
of  a  rabbit  is  sometimes  fatal.  Two  or  three  blood-agar  cultures  emulsified 
in  broth  should  be  used.  Under  these  conditions  the  organism  does  not 
generally  multiply  in  the  tissues  but  the  animal  dies  from  the  effects  of  the 
soluble  products  inoculated  at  the  same  time  as  the  organism. 

This,  and  the  fact  that  cultures  killed  with  chloroform  are  equally  fatal,  tends  to 
show  that  death  is  the  result  of  an  intoxication  and  not  of  an  infection.  Pfeiffer 
never  obtained  in  any  species  of  animal  but  monkeys  "  a  multiplication  of  the 
inoculated  bacilli,  that  is,  a  true  infection." 

It  is  nevertheless  possible  to  produce  an  infection  in  rabbits.  Thus, 
Meunier  and  also  Elmassian  produced  a  fatal  infection  in  rabbits  by  intra- 


506  PFEIFFER'S   BACILLUS 

venous  inoculation  and  were  able  to  demonstrate  a  multiplication  of  the 
bacilli  in  the  blood  and  in  the  pulmonary  and  renal  lesions. 

Kruse  produced  sub-cutaneous  abscesses  and  found  living  bacilli  in  the 
pus.  Slatineano,  and  Delius  and  Kolle  infected  rabbits  by  intra-peritoneal 
inoculation.  Cantani,  Slatineano,  and  Martin,  set  up  a  fatal  disease  by  inocu- 
lating small  doses  of  culture  into  the  brain. 

Mixed  infections. — Rosenthal  inoculated  a  mixture  of  Pfeiffer's  bacillus  and  a 
non- virulent  staphylococcus  aureus  into  rabbits'  lungs  :  the  animals  died  "  of  pul- 
monary congestion  generally  accompanied  by  septicaemia."  Jacobson  produced  a 
fatal  infection  accompanied  by  generalization  of  the  bacillus  by  inoculating  rabbits 
intra-venously  with  a  mixture  of  streptococci  and  Pfeiffer's  bacillus. 

Guinea-pigs. — Guinea-pigs  are  almost  insusceptible  but  can  be  infected 
by  intra-peritoneal  inoculation  of  very  virulent  cultures  (Delius  and  Kolle, 
Elmassian,  Cantani  and  others). 

Mice. — Mice  die  from  toxaemia  after  intra-peritoneal  inoculation  of  large 
doses  of  culture.  Infection  may  also  be  produced  by  intra-peritoneal  inocula- 
tion of  small  doses  of  virulent  cultures,  or  by  mixing  the  bacillus  with  a 
sterilized  culture  of  streptococci  (Jacobson). 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

Pfeiffer's  bacillus  is  a  very  small  rod-shaped  organism  with  rounded  ends, 
having  practically  the  appearance  of  a  cocco-bacillus.  It  is  one  of  the  smallest 
visible  micro-organisms,  is  non-motile,  and  occurs  singly  or  arranged  in  small 
chains  composed  of  two  to  four  bacilli.  In  sputum  the  bacilli  are  often  seen 
massed  together  in  large  numbers.  The  organism  is  sometimes  found  within 
the  leucocytes.  In  cultures,  it  is  a  little  larger  and  longer  than  in  sputum. 


«• 


FIG.  243.—  Pfeiffer's  bacillus—  Sputum—  Dilute  FIG.  244.—  Pfeiffer's  bacillus.    Film  from 

carbol-fuchsin.    (Reich,  oc.  IV,  obj.  ^th.)  a  blood-agar  culture.  Dilute  carbol-fuchsin. 

(Reich,  oc.  IV,  obj.  TUh.) 

Klein  lays  stress  upon  the  frequency  with  which  long  strepto-bacillary  filaments 
and  bacilli  with  a  central  vacuole  are  seen  in  cultures.  He  has  also  noticed  involu- 
tion forms  :  long  sinuous  bacilli  sometimes  looking  like  a  tangled  mass  of  filaments, 
as  well  as  large  oval  or  club-shaped  bacilli. 

Staining  reactions.  —  Pfeiffer's  bacillus  does  not  stain  very  readily  with 
the  basic  aniline  dyes  and  is  gram-negative.  The  best  method  is  to  stain  with 
dilute  carbol-fuchsin  for  about  10  minutes.  Carbol-methylene  blue,  carbol- 
thionin  or  Nastikow's  violet  may  also  be  used.  Staining  may  be  accelerated 
by  heating  the  dye. 


MORPHOLOGY  507 

Sections. — Sections  through  influenzal  broncho-pneumonic  patches  contain 
numerous  bacilli.  Pfeiffer  recommends  the  following  method  for  staining  the 
bacilli  in  sections  : 

Fix  in  alcohol,  embed  in  celloidin  (paraffin  is  better,  Besson)  stain  for 
half  an  hour  in  dilute  carbol-fuchsin,  differentiate  for  a  few  seconds  in 
absolute  alcohol  slightly  acidified  with  acetic  acid — the  deep  red  colour  of 
the  sections  is  now  changed  to  a  uniform  pinkish- violet  tint — clear  in  clove 
oil  and  xylol  and  mount  in  balsam.  The  bacilli  are  stained  deep  red  and  the 
ground  work  a  pale  pink. 

2.  Cultural  characteristics. 

Conditions  of  growth. — Pfeiffer's  bacillus  does  not  grow  on  the  ordinary 
media  :  for  its  cultivation  outside  the  body  media  containing  blood,  serum, 
or  haemoglobin  must  be  used.  It  is  strictly  aerobic  and  grows  at  temperatures 
between  26°  and  42°  C.,  the  optimum  being  37°  C. 

When  an  emulsion  of  sputum  containing  Pfeiffer's  bacillus  is  sown  on  agar  (p.  192) 
a  scanty  growth  of  the  organism  is  generally  obtained  but  sub-cultures  on  the  same 
medium  fail.  The  sputum  apparently  supplies  the  substances  necessary  for  the 
growth  of  the  primary  culture ;  but  for  sub-cultures  blood-agar  must  be  used. 
It  is  important  that  the  agar  should  be  neutral  or  only  just  alkaline,  for  even  a 
slight  excess  of  alkali  may  give  a  negative  result. 

Characters  of  growth.  Blood-agar. — Agar  to  which  blood  has  been  added 
is  the  best  medium  for  growing  Pfeiffer's  bacillus.  To  prepare  the  medium, 
liquefy  a  tube  of  agar  and  pour  the  contents  into  a  Petri  dish  :  when  set 
spread  a  large  drop  of  blood  in  as  thin  a  layer  as  possible 
over  the  surface.  Agar  slopes  may  be  treated  in  the 
same  way.  Human  blood  or  the  blood  of  rabbits  or 
pigeons,  collected  of  course  aseptically,  gives  the  best 
results.  Blood-agar  prepared  according  to  Bezan9on's 
directions  (p.  53)  is  equally  suitable. 

Surface  cultures  on  blood-agar  sown  with  an  emulsion 
of  sputum  and  incubated  at  37°  C.  for  24-48  hours  give 
a  copious  growth  of  very  small,  delicate  colonies  like  245  _Pfeiffer»s 

minute  dew  drops,  visible  only  with  a  lens  :  the  colonies  bacillus.  Single  colonies 
never  become  confluent.  A  colony  here  and  there  may  at  3^cd)agax handYeS8 
attain  the  size  of  a  pin's  head. 

Hsemoglobin-agar. — Huber  relying  on  Pfeiffer's  observation  that  haemo- 
globin is  the  constituent  in  blood  essential  for  the  growth  of  the  bacillus 
prepared  an  haemoglobin  medium. 

Huber  used  Hommel's  commercial  haemoglobin  :  this  liquid,  deep  red  in  colour, 
was  made  strongly  alkaline  with  potash  solution  to  prevent  coagulation  during 
heating  and  then  sterilized  at  100°  C.  The  product  was  added  to  sterile  melted 
agar  cooled  to  50°  or  60°  C.  in  sufficient  quantity  to  make  the  agar  deep  red  in 
colour.  The  tubes  were  then  sloped  and  allowed  to  set.  The  medium  is  not  very 
satisfactory,  and  the  same  remark  is  applicable  to  Nastikow's  method  of  adding 
yolk  of  egg  to  agar. 

It  is  better  to  use  a  1  per  cent,  aqueous  solution  of  commercial  haemoglobin  and 
to  sterilize  by  filtration  through  a  porcelain  bougie  (Achalme,  Rosenthal). 

Liquid  media. — In  broth  containing  pigeons'  blood  Pfeiffer's  bacillus  give& 
rise  to  delicate  whitish  flakes  but  the  growth  is  not  particularly  charac- 
teristic. A  better  cultivation  is  obtained  by  using  rabbit  serum  into  which 
a  certain  amount  of  haemoglobin  has  been  allowed  to  diffuse  by  leaving  the 
serum  in  contact  with  the  clot  (Rosenthal). 

Pfeiffer's  bacillus  does  not  grow  on  glycerin-agar.     The  cultures  which  were 


508  PFEIFFER'S   BACILLUS 

obtained  by  Kitasato  and  by  Bruschettini  and  Canon  were  not  cultures  of  Pfeiffer's 
bacillus. 

Ascitic  fluid  media  are  not,  as  a  rule,  of  much  use  for  growing  Pfeiffer's  bacillus 
though  very  suitable  for  Elmassian's  micro-organism. 

SECTION  III.— BIOLOGICAL   PROPERTIES. 
1.  Vitality  and  Virulence. 

Pfeiffer's  bacillus  is  very  sensitive  to  heat  and  drying.  In  sputum  which 
is  kept  moist  the  organism  retains  its  vitality  for  14  days  ;  but  if  allowed  to 
dry  at  ordinary  temperatures,  the  organism  is  killed  in  36  hours.  Desicca- 
tion at  37°  C.  sterilizes  cultures  of  the  bacillus  in  2  hours  and  at  ordinary 
temperatures  in  24  hours. 

In  cultures  on  blood-agar,  the  bacillus  lives  for  a  week  to  a  fortnight ;  and 
by  sub-cultivating  on  this  medium  every  4  or  5  days  it  can  be  kept  alive  for 
several  months.  In  a  collodion  sac  in  the  peritoneal  cavity  of  a  rabbit  the 
organism  lived  for  nearly  2  months  (Dujardin-Beaumetz). 

The  virulence  of  Pfeiffer's  bacillus  is  subject  to  considerable  variation. 
By  mixing  the  organism  with  streptococci  and  passing  the  mixture  through 
mice  the  virulence  can  be  raised  for  a  short  period  but  not  for  long. 

2.  Toxin. 

Cultures  killed  with  chloroform  or  heat  are  toxic.  Heated  cultures  inocu- 
lated into  the  cerebro-spinal  fluid  of  rabbits  killed  the  animals  in  2-3  hours 
(Martin  and  Dujardin-Beaumetz). 

Slatineano  prepared  an  endo-toxin  as  follows : 

Cultures  on  blood-agar  24  hours  old,  either  living  or  killed  by  heat  at  55°  C. 
were -made  into  an  emulsion  with  normal  saline  solution,  centrifuged  and  the  deposit 
dried.  0*25  gram  of  dried  bacilli  were  mixed  with  10  c.c.  of  a  mixture  of  equal 
parts  of  distilled  water  and  normal  horse  serum,  left  in  the  ice  chest  for  12  hours 
and  then  centrifuged. 

The  supernatant  liquid,  in  doses  of  O045  c.c.,  inoculated  into  the  brain  of 
guinea-pigs,  proved  fatal  in  6-10  hours,  and  when  given  in  quantities  of  5  c.c. 
into  the  peritoneal  cavity  the  animal  died  in  a  few  days. 

3.  Immunity.     Serum  therapy. 

Kolle  and  Delius  failed  to  immunize  animals  against  Pfeiffer's  bacillus 
but  Slatineano,  Cantani,  and  Latapie,  obtained  more  promising  results. 

Cantani  inoculated  guinea-pigs  sub-cutaneously  with  increasing  doses  of  very 
virulent  cultures  on  blood-agar  sterilized  at  56°  C.  One  guinea-pig  was  inoculated 
with  181  cultures  over  a  period  of  4  months.  The  animals  were  then  tested  by 
inoculating  many  times  the  minimal  lethal  dose  into  the  peritoneal  cavity.  Many 
of  the  animals  did  not  survive  the  immunizing  process,  but  those  which  did  proved 
to  be  highly  immune  and  were  able  to  resist  the  intra- peritoneal  inoculation  of  100 
lethal  doses.  The  serums  of  the  immunized  animals  exhibited  prophylactic  pro- 
perties in  different  degrees  :  the  best  results  were  obtained  when  the  test  animal  was 
inoculated  with  the  serum  of  an  immunized  animal  mixed  with  a  virulent  culture. 

Latapie  immunized  a  goat  by  inoculating  it  first  with  dead  then  with 
living  cultures  of  a  strain  of  Pfeiffer's  bacillus  isolated  from  a  case  of  influenza  ; 
several  inoculations  were  given  and  the  immunizing  process  was  extended  over 
a  year.  The  animal  was  bled  1  month  after  the  last  inoculation,  and  tho 
serum  was  found  to  be  capable  of  protecting  guinea-pigs  against  two  or  three 
fatal  doses  of  the  bacillus,  provided  that  it  was  inoculated  in  doses  of  1-3  c.c. 
either  sub-cutaneously  several  hours  before,  or  intra-venously  a  few  hours 
before,  the  test  inoculation. 


SECONDARY  INFECTIONS  509 

4.  Agglutination. 

Pfeiffer's  bacillus  is  not  specifically  agglutinated  by  the  serum  of  patients 
suffering  from  influenza  and  in  whose  sputum  the  organism  is  present 
(Meunier).1 

The  serum  of  immunized  animals  agglutinates  the  bacillus  in  dilutions  of 
1  in  200  to  1  in  500  (Cantani). 

5.  Secondary  infections— Ancillary  micro-organisms. 

In  persons  suffering  from  influenza  certain  micro-organisms  are  able  to 
develop  side  by  side  with  Pfeiffer's  bacillus 2  and  produce  complications 
of  the  original  disease.  The  micro-organisms  commonly  found  associated 
with  Pfeiffer's  bacillus  are  streptococci.,  pneumococci,  staphylococci  and  bacillus 
coli.  These  micro-organisms  appear  to  favour  the  development  of  Pfeiffer's 
bacillus  and  to  facilitate  its  growth. 

Grassberger  has  pointed  out  that  in  artificial  cultures,  in  presence  of  the 
staphylococcus  aureus,  the  colonies  of  Pfeiffer's  bacillus  grow  to  an  unusually 
large  size  so  that  they  become  visible  to  the  naked  eye  in  24  hours. 

The  colonies  remain  transparent,  and  nearly  colourless  or  bluish,  the  centres  being 
sometimes  greyish  in  colour :  they  are  almost  confluent  but  their  margins  do  not 
blend.  These  giant  "  influenza  "  colonies  are  seen  lying  in  the  hollows  between  the 
larger  staphylococcal  colonies :  sub-cultivated  alone  on  blood-agar  (without  the 
staphylococci)  they  give  rise  to  colonies  of  normal  appearance  which  retain  their 
vitality  longer  than  usual. 

Pfeiffer's  bacillus  is  less  sensitive  also  to  the  reaction  of  the  culture  medium  when 
grown  symbiotically  with  the  staphylococcus,  so  that  it  will  then  grow  on  quite 
markedly  alkaline  blood-agar. 

The  various  staphylococci,  and  in  a  lesser  degree  the  colon  bacillus,  the 
typhoid  bacillus,  the  diphtheria  bacillus,  and  streptococci  all  exhibit  this 
ancillary  influence. 

The  influence  of  these  organisms  on  Pfeiffer's  bacillus  seems  to  depend  upon  the 
action  of  certain  substances  secreted  by  them  or  to  some  change  induced  in  the 
medium  as  a  result  of  their  growth.  Thus,  if  a  24-hour  agar  culture  of  a  staphy- 
lococcus be  sterilized  in  the  autoclave,  poured  into  a  Petri  dish,  and  sown  with 
Pfeiffer's  bacillus  after  smearing  the  surface  with  blood,  the  organism  will  grow 
in  the  form  of  the  large  colonies  referred  to  above  (Rosenthal). 

This  fact  can  be  easily  demonstrated  in  the  following  manner  :  Sow  a 
culture  of  Pfeiffer's  bacillus  all  over  the  surface  of  a  blood-agar  medium  and 
after  incubating  for  3  or  4  hours  at  37°  C.  to  evaporate  the  excess -of  moisture 
and  to  get  rid  of  bubbles,  sow  a  narrow  stroke  culture  of  the  staphylococcus 
on  the  surface  or  make  two  or  three  punctiform  stabs.  On  incubation  at 
37°  C.  Pfeiffer's  bacillus  will  form  giant  colonies  like  satellites  around  the 
staphylococcal  colonies.3 

Rosenthal  suggests  that  this  ancillary  action  of  the  staphylococcus  might 
be  applied  in  cases  where  search  for  Pfeiffer's  bacillus  has  yielded  doubtful 
results  :  thus  if  tubes  sown  with  sputum  or  blood  remain  sterile  after  incubat- 
ing for  24  hours,  he  suggests  that  a  staphylococcus  might  be  sown  in  a 
narrow  streak  on  the  culture  medium  in  the  hope  that  it  might  stimulate 
the  latent  bacillus. 

1  It  must  be  remembered  that  normal  human  serum  and  the  serum  of  normal  animals 
will  agglutinate  Pfeiffer's  bacillus  in  dilutions  of  1  in  10  to  1  in  20. 

2  In  6  only  out  of  30  cases  of  influenza  studied  by  Grassberger  was  Pfeiffer's  bacillus 
found  in  pure  culture. 

3  The  Micrococcus  prodigiosus  has  a  similar  effect,  but  the  cultures  of  this  coccus  must 
be  sterilized  by  heating  for  20  minutes  at  60°  C.,  since  sterilization  in  the  autoclave  destroys 
the  action  (Luerssen). 


510  PFEIFFER'S   BACILLUS 

According  to  Rosenthal,  Pfeiffer's  bacillus  exerts  a  reciprocal  ancillary  action  on 
the  pneumococcus  :  thus  after  sub-cultivating  a  pure  culture  of  the  latter  on  blood- 
agar  a  few  times  the  organism  dies  out,  but  if  it  be  mixed  with  Pfeiffer's  bacillus  the 
pneumococcus  may  be  sub-cultivated  almost  indefinitely  on  blood-agar. 


SECTION  IV.— THE   DETECTION  AND   ISOLATION   OF   PFEIFFER'S 

BACILLUS. 

In  cases  of  influenza  Pfeiffer's  bacillus  occurs  in  the  sputum  and  nasal 
mucus,  and  more  rarely  in  the  blood  ;  for  the  purpose  of  demonstrating 
the  bacillus  it  is  best  to  examine  the  sputum.  Pfeiffer  lays  stress  on  the 
somewhat  characteristic  appearance  of  the  sputum — thick,  purulent,  greenish- 
yellow  and  generally  inspissated  in  small  compact  masses  :  the  bacillus  lies 
between  and  within  the  pus  cells.  Microscopical  examination  should  always 
be  supplemented  by  cultivation.  In  sowing  the  sputum  certain  precautions 
are  necessary. 

Select  a  very  solid  portion  of  the  sputum,  and  after  washing  it  several 
times  in  sterile  distilled  water  (Kitasato's  method  :  p.  192)  remove  a  fragment 
from  the  centre  without  introducing  contaminations  and  sow  it  on  the  surface 
of  blood-agar  plates. 


THE  H^IMOGLOBINOPHILIC  BACILLI. 

1.  The  pseudo-influenza  bacillus  of  Pfeiffer,  distinguished  from  Pfeiffer's  influenza 
bacillus  by  its  rather  larger  size  and  by  its  property  of  forming  filaments  in  culture, 
is  now  admitted  by  Pfeiffer  and  others  to  be  identical  with  the  latter  organism  as 
was  suggested  many  years  ago  by  Besson. 

Grassberger's  micro-organisms  A  and  B  and  the  haemophilic  cocco-bacillus  of 
Rosenthal  must  also  be  regarded  as  identical  with  Pfeiffer's  bacillus. 

The  bacillus  found  by  Jochmann,  Krause  and  Moltrecht  in  the  sputum  and  in 
the  broncho-pneumonic  patches  of  cases  of  whooping  cough,  and  known  as  the 
B.  pertussis  Eppendorf,  should  in  the  absence  of  further  knowledge  and  if  the 
description  given  by  these  observers  be  accepted  also  be  considered  as  identical 
with  Pfeiffer's  bacillus. 

Elmassian's  bacillus  has  all  the  morphological  characteristics  of  Pfeiffer's  bacillus, 
but  is  highly  pathogenic  to  guinea-pigs  in  which  animals  it  produces  a  rapidly  fatal 
septicaemia.  Probably  this  organism  is  one  of  a  group  of  haemophilic  micro-organisms 
closely  related  to  Pfeiffer's  bacillus.  Friedberger's  B.  hcemoglobinophilus  canis 
and  the  bacillus  isolated  by  Wolff  from  the  bronchial  mucus  of  a  rat  which  had 
died  as  the  result  of  the  inoculation  of  cholera  toxin  are  members  of  the  same  group. 

2.  The  bacillus  of  acute  contagious  conjunctivitis. 

Acute  contagious  conjunctivitis  is  caused  by  a  bacillus  described  by  Weeks  and 
Morax  :  the  organism  is  very  similar  to  Pfeiffer's  bacillus. 

Jundell  thinks  that  the  conjunctivitis  due  to  Weeks'  bacillus  is  possibly  a  localized 
influenza  in  the  eye,  and  that  the  causal  organisms  in  the  two  diseases  are  merely 
variants  of  one  and  the  same  species. 

Experimental  inoculation. — Weeks'  bacillus  is  not  pathogenic  for  animals  when 
inoculated  on  the  conjunctiva.  In  man,  however,  a  trace  of  a  culture  smeared  on 
the  conjunctiva  gives  rise  to  an  acute  conjunctivitis. 

Morphology. — The  bacillus  occurs  as  small  very  slender  and  very  short  rods, 
non-motile,  arranged  either  singly  or  in  chains  of  two  or  three  bacilli,  either  free 
or  within  the  leucocytes.  In  cultures,  the  bacillus  often  assumes  an  elongated  form. 
It  stains  with  the  ordinary  aniline  dyes  and  is  gram-negative. 

Cultural  characteristics. — The  organism  is  aerobic  and  fails  to  grow  or  grows 
very  feebly  on  the  ordinary  media,  but  on  media  containing  blood  or  serum  gives 
rise  to  a  good  growth.  Growth  only  takes  place  at  37°  C.  The  cultural  charac- 
teristics are  in  every  way  similar  to  those  of  Pfeiffer's  bacillus. 


THE   BACILLUS   OF   WHOOPING  COUGH  511 

3.  The  bacillus  of  sub-acute  conjunctivitis. 

The  organism  described  by  Morax  is  a  large  diplo-bacillus,  measuring  2— 3/z  long 
and  about  I/A  broad.  It  is  non-motile  and  occurs  singly  or  grouped  in  clumps  or 
chains,  it  stains  with  the  basic  aniline  dyes  and  is  gram-negative.  In  the  conjunctival 
secretions,  the  bacilli  are  free  or  intra- cellular. 

Morax' s  bacillus  is  aerobic  and  grows  only  on  media  con  taming  blood  or  serum. 
Cultures  retain  their  vitality  for  a  long  time  at  37°  C.,  but  are  killed  in  15  minutes 
at  a  temperature  of  58°  C. 

A  drop  of  culture  placed  in  the  conjunctival  reflection  in  man  sets  up  a  typical 
conjunctivitis.  The  bacillus  has  no  pathogenic  action  on  the  conjunctiva  of  animals. 


THE  BACILLUS  OF  WHOOPING  COUGH  (Bordet-Gengou). 

Numerous  micro-organisms  have  from  time  to  time  been  isolated  from  the 
bronchial  secretions  of  children  suffering  from  whooping  cough.  These  may  be 
regarded  as  belonging  to  one  of  two  types :  (1)  bacilli  which  only  grow  on  media 
containing  haemoglobin  and  which  must  be  considered  as  identical  with  Pfeiffer's 
bacillus,  e.g.  the  organism  isolated  by  Jochmann  and  Krause  (p.  510) :  (2)  bacilli 
which  grow  on  ordinary  media  e.g.  the  bacilli  isolated  by  Afanassiew,  Vincenzi, 
Czaplewski  and  Henger,  etc.  In  the  case  of  none  of  these  organisms  has  proof 
been  adduced  in  support  of  their  specific  relationship  to  the  disease. 

Bordet  and  Gengou,  on  the  other  hand,  have  brought  forward  convincing 
arguments  in  favour  of  the  specificity  of  an  organism  which  they  isolated 
from  the  sputum  of  cases  of  whooping  cough.  Its  appearance  and  charac- 
teristics are  as  follows  : 

1.  Morphology. — A  small  bacillus  often  assuming  a  cocco-bacillary  form, 
staining  more  deeply  at  the  ends  than  in  the  centre,  gram-negative,  occasion- 
ally intra-cellular   and   exhibiting  pleomorphic   features   in    liquid    culture 
media. 

2.  Cultural  characteristics. — The  organism  grows  feebly  and  cannot  be 
cultivated  on  the  ordinary  media  either  under  aerobic  or  anaerobic  conditions. 
It  grows  best  on  an  agar  medium  made  with  a  1  per  cent,  glycerin-potato 
mash  to  which  an  equal  volume  of  human  or  rabbit  blood  or  serum  (haemo- 
globin is  not  essential)  has  been  added. 

Primary  cultures  are  difficult  to  obtain  and  the  growth  is  always  very 
scanty,  so  scanty  indeed  as  often  to  be  invisible  to  the  naked  eye.  By  sub- 
cultivating  after  incubating  for  48  hours  at  37°  C.  a  better  growth  is  obtained, 
consisting  of  a  narrow,  whitish,  slightly  raised  but  distinctly  visible  streak 
which  becomes  thicker  and  whiter  in  subsequent  sub-cultures.  Growth  is 
then  easily  obtained  on  ascitic-agar,  serum-broth,  blood-broth,  etc. 

3.  Detection  and  isolation  of  the  bacillus. — The  bacillus  of  Bordet-Gengou 
should  be  sought  for  in  the  early  stages  of  the  disease  when  the  fits  of  coughing 
first  begin  ;    at  a  later  period  its  isolation  becomes  difficult  or  impossible  on 
account  of  the  numerous  other  organisms  which  have  become  mixed  with  it. 
To  isolate  the  organism  sow  a  fragment  of  the  bronchial  exudate,  not  mixed 
with  saliva,  on  the  surface  of  the  special  blood-potato-agar. 

4.  The  following  facts  constitute  the  evidence  upon  which  the  organism  is  claimed 
to  be  the  cause  of  the  disease. 

a.  The  bacillus  of  Bordet  and  Gengou  is  agglutinated  by  the  serum  of  children 
who  have  recently  recovered  from  whooping  cough,  though  not  in  every  case :  it  is 
however  always  agglutinated  by  the  serum  of  an  immunized  horse,  even  in  a  dilution 
of  1  in  5,000. 

6.  The  serum  of  children  who  have  recovered  from  the  disease  always  contains 
a  specific  immune  body  (sensibilisatrice]  the  presence  of  which  was  proved  by  Bordet 
and  Gengou  by  the  method  of  complement  fixation. 


512  THE   BACILLUS   OF  WHOOPING   COUGH 

c.  The  bacillus  of  Bordet  and  Gengou,  when  inoculated  into  the  peritoneal  cavity 
of  a  guinea-pig,  causes  the  death  of  the  animal  from  toxaemia  :  the  endotoxin  of  the 
bacillus  sets  up  necrosis.  Klimenko  failed  to  reproduce  whooping  cough  in  guinea- 
pigs  and  most  laboratory  animals  :  but  in  young  dogs  and  in  monkeys  (Cynocephalus, 
Macacus,  Cercopithecus)  a  disease  characterized  by  fever,  catarrh  of  the  nasal  and 
ocular  mucous  membranes,  sneezing,  and  accompanied  by  a  hoarse  cough,  without 
spasms  but  occasionally  causing  vomiting,  followed  infection  by  the  bacillus  whether 
produced  by  inoculation  into  the  naso-pharyngeal  cavities  or  by  contagion.  Young 
dogs  often  died  of  pneumonia  after  about  5  or  6  weeks  and  the  bacillus  was  found  in 
the  laryngeal  secretion  and  in  the  pulmonary  foci. 


CHAPTER  XXXIV. 
THE   BACILLUS  OF  SOFT  SORE. 

Introduction. 

Section  I. — Experimental  infection,  p.  513. 

Section  II. — Morphology  and  cultural  characteristics,  p.  514. 

Section  III. — Biological  properties,  p.  515. 

Section  IV. — Detection,  isolation  and  identification  of  the  bacillus,  p.  515. 

THE  bacillus  of  soft  sore  was  discovered  by  Ducrey  and  more  completely 
studied  by  Unna,  Ch.  Nicolle,  Queyrat,  Bezan9on  and  others. 

In  pus  from  the  chancre  the  bacillus  is  generally  found  in  association  with 
other  organisms  (staphylococci,  micrococcus  tetragenus,  bacillus  cutis  communis  of 
Ch.  Nicolle  and  gonococci).  The  bacillus  is  found  between  the  cells  of  the  connective 
tissue  of  the  dermis,  but  does  not  penetrate  into  the  fixed  ceUs  of  the  tissues  nor 
gain  access  to  the  vessels ;  when  the  lesion  is  healing  numerous  bacilli  will  be  found 
in  the  leucocytes.  The  pus  becomes  less  and  less  pathogenic  as  the  sore  cicatrizes. 

The  bacillus  is  present  in  pure  culture  in  the  bubo,  but  as  a  rule  (92  times  out  of 
100,  Rille)  cannot  be  detected  on  microscopical  examination  of  the  pus — the  sterile 
pus  of  Strauss — though  its  presence  there  can  be  demonstrated  by  sowing  cultures 
(Bezan§on,  Griffon  and  Le  Sourd,  Simon). 

SECTION  I.— EXPERIMENTAL  INFECTION. 

Man. — If  a  little  pus  from  a  chancre  or  a  culture  of  the  bacillus  be  inocu- 
lated a  typical  soft  sore  is  produced  in  the  human  subject.  A  first  infection 
confers  no  immunity,  and  the  sore  is  indefinitely  re-inoculable  time  after 
time  in  the  same  individual. 

Inoculation  for  experimental  purposes  should  be  made  into  the  outer  surface  of 
the  arm  or  into  the  thighs  but  not  on  to  the  abdomen  below  the  umbilicus. 

It  is  best  to  lightly  scarify  the  epidermis  over  an  area  of  2  or  3  mm.  with  a  sterilized 
needle  charged  with  the  virus  ;  the  skin  should  be  scratched  sufficiently  deeply  to 
draw  a  drop  of  blood.  The  site  of  inoculation  should  be  protected  from  friction  and 
from  all  source  of  contamination :  the  most  convenient  method  of  doing  this,  and 
one  which  allows  of  the  lesion  being  watched,  is  to  cover  the  part  with  a  sterile 
watch-glass  which  can  be  held  in  position  by  a  piece  of  gauze  in  the  centre  of  which  a 
hole  is  cut  so  that  the  glass  is  only  covered  at  its  edges ;  the  gauze  is  fixed  to  the 
glass  and  to  the  skin  with  collodion.  The  whole  is  covered  with  a  little  wool  and 
bandaged.  The  lesion  commences  to  develop  about  the  end  of  the  first  day  and  is 
characteristic  about  the  fourth  to  the  sixth  day. 

Animals. — Laboratory  animals  are  immune  but  monkeys  (Macacus  and 
Bonnet  monkeys)  may  be  infected  experimentally.  Tomasczewski  has 
successfully  inoculated  man  with  cultures  from  an  experimental  sore  in 
a  monkey. 

2K 


514  THE   BACILLUS   OF   SOFT   SORE 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  organism  is  a  rather  short  bacillus  with  rounded  ends  measuring  O5// 
by  1*5  to  2/j.  Two  lateral  notches  are  sometimes  seen  which  give  the  organism 
a  figure-of-eight  appearance.  In  pus  it  occurs  singly  or  in  a  chain  composed 

of  three  to  twenty  bacilli,  and  clumps  may 
be    formed    of   chains    crowded   one    against 

^s     v  another.     The  bacilli  are  generally  free,  but 

i  xx  it  is  not  uncommon  to  find  them  inside  the 

polynuclear   leucocytes.     In   scrapings   from 
some  soft  sores  the  bacillus  does  not  show  its 
V^       ordinary  characteristics  but  assumes  the  form 
of  a  coccus  (Queyrat). 

In  cultures  on  blood-agar  the  bacilli  occur 
singly  or  collected  together  in  masses  or  in 
short  chains :  in  liquid  serum  they  form 
long  wavy  chains  arranged  like  a  skein  of  silk 
(streptobacilli)  :  in  the  liquid  at  the  bottom 
of  blood-agar  tubes  the  bacilli  are  more  slender 
?  and  the  chains  much  longer. 

Staining  reactions. — The   bacillus   of   soft 

sore  is  easily  stained  with  the  basic  aniline  dyes,  but  in  the  majority  of 
cases  only  the  ends  take  up  the  dye  leaving  the  centre  unstained  ( shuttle  - 
shaped  bacilli).  The  bacillus  is  gram-negative. 


t 

Off  * 
9 

»&          6 


g«r*jS* 

FIG.  247. — Bacillus  of  soft  sore.     Film  FIG.  248. — Bacillus  of  soft  sore.     Film 

from  a  culture  on  blood-agar.     (After  Be-  from  a  liquid  serum  culture.     (After  Bezan- 

zancon,  Griffon  and  Le  Sourd.)  con,  Griffon  and  Le  Sourd.) 

(a)  Pus  and  cultures. — Pus  and  cultures  should  be  stained  with  carbol- 
thionin,  carbol-blue  or  carbol- violet  and  examined  in  water  or  balsam. 

Krefting  recommends  the  following  solution  : 

5  per  cent,  solution  of  boric  acid  in  water,       -  16  c.c. 

Saturated  aqueous  solution  of  methylene  blue,  20     ,, 

Distilled  water,    -  24     „ 

Queyrat  recommends  the  following  mixture  for  smear  preparations  : 
Carbol-fuchsin,     -  10  drops. 

Saturated  aqueous  solution  of  methylene  blue,  7       „ 

Distilled  water,    -  .-__..  20  c.c. 

(b)  Sections. — The  method  recommended  for  staining  sections  is  Nicolle's 
tannin  method  (p.  217).     Stain  in  Kiihne's  carbol-blue  then  treat  for  a  few 
seconds  with  10  per  cent,  tannin  solution,  wash  in  water,  then  in  alcohol, 
clear  in  clove  oil  and  xylol  and  mount  in  balsam. 


BIOLOGICAL  PROPERTIES  515 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  bacillus  of  soft  sore  does  not  grow  on  the 
ordinary  media. 

It  would  seem  that  Petersen  was  able  occasionally  to  grow  the  bacillus  on  serum- 
agar  but  his  investigations  have  not  been  confirmed.  Istamanoff  and  Akspiantz 
as  well  as  Lenglet  employ  macerations  of  human  skin  solidified  with  agar,  but  their 
technique  is  obscure  and  their  results  unconvincing.  The  cultivation  of  the  bacillus 
has  been  made  practicable  by  the  investigations  of  Bezangon,  Griffon  and  Le  Sourd. 

The  most  suitable  medium  is  the  rabbit-blood-agar  of  Bezanyon  and  Griffon 
(p.  53)  and  after  that  liquid  rabbit-serum.  All  attempts  to  grow  the  organism 
on  the  ordinary  media  failed  even  after  acclimatizing  the  bacillus  by  sub- 
cultivating  it  on  a  series  of  blood-agar  tubes. 

The  bacillus  is  aerobic  and  grows  at  37°  C. 

Blood-agar. — After  sowing  surface  cultures  freely  with  pus  from  the  sore 
and  incubating  at  37°  C.  for  24  hours  colonies  appear  which  are  "  rounded, 
raised,  lustrous,  and  attain  their  maximum  size  in  48  hours  ;  they  are  then 
greyish  opaque  and  about  1-2  mm.  in  diameter. ' '  When  the  growth  is  removed 
for  microscopical  examination  it  has  a  tendency  to  slip  away  from  the  needle 
and  is  difficult  to  break  up  on  a  slide. 

Sometimes  the  colonies  only  appear  after  incubating  for  48  hours  and  are 
few  in  number.  The  growth  is  more  abundant  in  sub-cultivations,  in  which 
the  colonies  are  very  numerous  and  may  attain  the  size  of  a  pin's  head,  but 
they  never  coalesce  to  form  a  continuous  layer. 

Rabbit-serum.— Growth  is  poorer  in  liquid  serum  than  on  the  above  medium. 
The  serum  becomes  slightly  turbid  and  shows  a  few  little  flocculi  floating  in  it. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 

Vitality  and  virulence. — Pus  from  the  sore  will  remain  virulent  for  some 
time  if  kept  away  from  the  air  ;  Ricord  obtained  positive  results  after  inocu- 
lating pus  17  days  old.  The  virulence  is  preserved  equally  well  in  urine, 
water,  vaginal  mucus,  etc.  Drying  at  ordinary  temperatures  seems  to  destroy 
the  virulence  of  the  pus  in  24—36  hours  ;  heating  for  18  hours  at  37°  C.  or 
for  an  hour  at  42°  C.  also  destroys  the  virulence  (Aubert),  but  a  temperature 
of  -16°  C.  has  no  effect  on  it  (Jullien).  The  bacillus  is  quickly  destroyed  by 
weak  antiseptic  solutions  and  by  acids  and  alkalis. 

In  cultures  on  blood-agar  the  bacillus  retains  both  its  vitality  and  its 
virulence  for  a  very  long  time  :  sub-cultures  sown  from  cultures  kept  in  the 
incubator  at  37°  C.  for  more  than  3  weeks  give  positive  results,  and  after 
eleven  sub-cultures  on  blood-agar  the  bacillus  will  still  give  rise  to  a  soft 
chancre  in  man.  But  in  cultures  on  liquid  serum  the  vitality  of  the  organism 
is  of  short  duration  (Bezan9on,  Griffon  and  Le  Sourd). 


SECTION  IV.     DETECTION  ISOLATION  AND   IDENTIFICATION  OF 

THE   BACILLUS. 

To  determine  the  presence  of  the  bacillus  in  the  tissues  microscopical 
examination  should  be  supplemented  by  cultivation  experiments  and  by 
inoculation. 

(a)  Microscopical  examination.— Scrapings  from  the  sore. — Wipe  away  the 
pus  from  the  surface  of  the  sore,  lightly  scrape  the  base  of  the  ulcer  with  a 
strong  platinum  needle  and  spread  the  material  on  slides,  taking  care  not  to 


516  THE  BACILLUS   OF  SOFT  SORE 

treat  the  preparation  roughly  otherwise  the  chains  will  be  broken  up.     Dry, 
fix  and  stain  (vide  ante). 

Sections. — Fix  small  pieces  of  skin  in  acid  perchloride,  harden  in  alcohol 
and  embed  in  paraffin.     Stain  according  to  the  method  given  above. 

(b)  Cultures. — Sow  tubes  of  blood-agar  with  pus  from  the  chancre  or  bubo, 
using  a  liberal  amount  of  material.     To  collect  the  material  for  sowing  cul- 
tures allow  the  pus  to  accumulate  beneath  a  dry  dressing,  which  should  be 
applied  to  the  sore  after  disinfecting  the  latter  with  tincture  of  iodine. 

(c)  Inoculations. — Inoculation  if  made  at  all  should  be  made  on  the  man 
infected  with  the  chancre  under  investigation  (p.  513).     In  this  way  the  nature 
of  the  sore  may  in  some  cases  be  determined,  for  if  the  inoculation  "  take," 
the  experiment  indicates  that  the  chancre  is  a  soft  sore.     A  positive  result 
does  not  however  exclude  the  possibility  of  a  co-existent  syphilitic  infection  ; 
and  soft  sores  are  often  due  to  a  mixed  infection  with  the  bacillus  of  soft 
sore  and  the  Treponema  pallidum. 


CHAPTER  XXXV. 
.    BACILLUS   ANTHRACIS. 

Introduction. 

Section  I. — The  experimental  disease,  p.  518. 

1.  Susceptible  and  immune  animals,  p.  518.     2.  Methods  of  inoculation,  p.  519. 
3.  Symptoms  and  lesions  in  experimental  animals,  p.  519. 
Section  II. — Morphology,  p.  520. 

1.  Microscopical  appearance  and  staining  reactions,  p.  520.     2.  Cultural  charac- 
teristics, p.  524. 
Section  III. — Biological  properties,  p.  525. 

1.  Viability  and  resistance,  p.   525.     2.  Virulence,   Attenuation,  Pasteur's  vac- 
cination, p.   527.     3.  Toxin,  p.   529.     Vaccination  with  toxin,  p.  530.     4.  Serum 
therapy,  p.  530.     5.  Agglutination,  p.  533. 
Section  IV. — Detection,  isolation  and  identification  of  the  anthrax  bacillus,  p.  533. 

Distribution  of  the  bacillus,  p.  533.  Methods  of  examination,  p.  534. 
Examination  of  carcases  dead  of  anthrax,  p.  534.  Isolation  of  the  bacillus  from 
soil,  p.  535. 

THE  anthrax  bacillus  is  the  cause  of  anthrax  in  man  and  the  lower  animals. 

In  man  three  forms  of  the  disease  are  commonly  recognized  viz.  malignant  pustule, 
pulmonary  anthrax  or  woolsorter's  disease,  and  intestinal  anthrax.  These  three 
clinical  types  correspond  to  the  three  channels  of  infection.  The  skin  infection 
(malignant  pustule)  follows  contamination  of  an  abraded  surface  when  handling 
the  hides  or  flesh  of  animals  dead  of  the  disease.  [In  England  malignant  pustule 
is  seen  chiefly  among  the  hide  porters  at  the  various  ports,  and  in  the  tanneries 
of  South  London.  ]  Infection  of  the  respiratory  passages  results  from  the  inhala- 
tion of  dust  containing  the  spores  of  the  bacillus ;  [it  is  the  characteristic  infection 
in  the  wool- combing  sheds  of  Yorkshire]  and  is  hence  sometimes  known  as  the 
Bradford  disease.  Intestinal  anthrax  is  very  uncommon  ;  when  it  occurs  it  is  due 
to  the  consumption  of  meat  from  anthrax-infected  animals. 

In  the  lower  animals  the  disease  is  variously  designated  :  the  commonest  descrip- 
tion being  "  splenic  fever  "  or  "  splenic  apoplexy  " — of  sheep  and  cattle.  Anthrax 
in  cattle  and  pigs  in  Brazil  is  known  as  garotilho. 

Infection  of  domestic  animals  takes  place,  as  a  rule,  via  the  alimentary  canal  by 
means  of  food  contaminated  with  the  spores  of  the  bacillus.  When  anthrax-infected 
carcases  are  buried  the  bacilli  which  are  very  numerous  in  the  blood  form  spores. 
These  are  brought  to  the  surface  in  worm  casts  and  being  washed  over  the  ground  by 
rain  are  subsequently  taken  up  by  animals  grazing  over  the  infected  area,  and 
should  the  epithelial  lining  of  the  alimentary  canal  be  abraded  by  thorns,  splinters 
of  wood  or  other  similar  substance  present  in  the  food-stuff,  the  spores  find  a  point 
of  entry  into  the  tissues  and  multiplying  under  the  favourable  conditions  of  their 
environment  give  rise  to  the  disease  (Pasteur).  In  Brazil,  the  carcases  of  anthrax-, 
infected  animals  are  devoured  by  vultures,  and  the  latter  disseminating  the  spores 
in  their  excreta  are,  according  to  March oux  and  Salimbeni,  the  chief  agents  in  the 
spread  of  garotilho. 


518  THE   ANTHRAX   BACILLUS 

In  cases  of  spontaneous  infection  in  man  and  the  lower  animals  it  may  be 
stated  that  the  less  severe  the  local  reaction  the  more  severe  the  subsequent 
septicaemia.  Malignant  pustule  in  man,  where  the  most  prominent  feature 
is  the  external  lesion,  only  exceptionally  leads  to  death  [ — according  to 
figures  given  by  J.  M.  Legge  the  death  rate  from  cutaneous  anthrax  is 
nevertheless  about  24  per  cent,  in  this  country  (61  deaths  among  255  cases 
in  six  years)].  On  the  other  hand  in  domestic  animals  the  local  reaction  is 
almost  nil  (sometimes  glosso-anthrax),  and  death  usually  supervenes. 


SECTION  I.— THE  EXPERIMENTAL  DISEASE. 
1.  Susceptible  and  immune  animals. 

1.  Sheep. — In  sheep  the  disease  runs  a  very  rapid  course  :  death  often  takes 
place  suddenly  following  an  attack  of  hsemoglobinuria.     Generally  speaking 
this  species  is  highly  susceptible  to  infection  both  by  sub-cutaneous  inocula- 
tion and  by  ingestion  ;    Algerian  sheep  however  are  immune  (Chauveau). 

2.  Rodents. — Mice,  guinea-pigs  and  rabbits  are  very  susceptible  to  sub- 
cutaneous inoculation  but  are  less  easily  infected  by  feeding. 

Rats  generally  show  a  higher  degree  of  immunity.  White  rats  are  in  most 
cases  immune  but  the  immunity  is  not  absolute  and  is  subject  to  great  varia- 
tion. Young  rats  are  more  susceptible  than  adults  of  the  species. 

Rats  may  be  inoculated  with  anthrax  bacilli  several  times  without  infecting 
them  though  it  is  always  possible  that  a  further  inoculation  may  produce  the 
disease  (Straus).  A  virus  virulent  for  adult  rats  can  be  obtained  by  passage 
through  young  white  rats  (Metchnikoff).  Overfeeding  lowers  the  resistance  of  the 
rat  and  renders  it  susceptible  to  infection  (Charrin  and  Roger).  Feser  believes  the 
immunity  to  be  more  constant  in  animals  fed  on  meat. 

Behring,  Metchnikoff  and  Roux,  and  Sawtehenko,  have  shown  that  the  serum  of 
white  rats  contains  a  lysin  capable  of  dissolving  the  anthrax  bacillus  in  vitro. 

3.  Bovine  animals. — Cattle  are  very  susceptible  to  infection  through  the 
intestinal  canal  but  are  more  resistant  to  sub-cutaneous  inoculation.     When 
infected  by  feeding  the  animal  is  attacked  with  a  blood-stained  diarrhoea, 
colic,  sweating  and  convulsions  and  dies  after  a  few  hours'  illness. 

4.  Horses. — The  horse  is  not  often  infected  through  the  intestinal  canal, 
though  intestinal  anthrax  occurs  as  an  epizootic  among  horses  in  Russia  and 
in  Corsica.     [The  epizootics  of  anthrax  in  Siberia  are  known   as   Siberian 
fever.]     Horses  are  more  susceptible  than  cattle  to  sub-cutaneous  inocula- 
tion. 

5.  Pigs. — These  animals  are  almost  entirely  immune  to  anthrax. 

Carini  has  recorded  the  occurrence  in  Brazil  of  an  anthrax  infection  of  pigs  accom- 
panied by  swelling  of  the  cervical  glands  (qarotilho),  but  has  been  unsuccessful  in  his 
attempts  to  reproduce  the  disease  experimentally. 

6.  Carnivora. — As  a  rule  these  animals  are  only  slightly  susceptible  to 
anthrax.     Bears  and  cats  seem  more  susceptible  than  other  animals  of  this 
family.     The  fox  is  immune  (Amler). 

Dogs  are  naturally  immune  to  anthrax  [but  spontaneous  infection  has  been 
observed  following  upon  the  consumption  of  infected  horse  meat].  Sub-cutaneous 
inoculation  generally  results  in  the  formation  of  an  abscess  in  which  active 
phagocytosis  takes  place  so  that  the  animal  escapes  a  generalized  infection.  The 
immunity  of  the  dog  may  be  overcome  by  inoculating  very  large  quantities  of  the 
virus  into  the  veins,  by  the  intra-venous  inoculation  of  an  emulsion  of  wood- 
charcoal,  by  extirpation  of  the  spleen,  etc.  Young  dogs  readily  succumb  to 
intra-pleural  inoculation  (Nocard).  A  mad  dog  inoculated  with  1  c.c.  of  a  culture 
harmless  to  a  healthy  dog  dies  of  anthrax  in  less  than  24  hours.  The  virus  thus 


EXPERIMENTAL   INFECTION  519 

obtained    is    considerably   increased    in   virulence   and   can   be    used    for    passage 
experiments  in  dogs  (Martel). 

7.  Birds. — Fowls  are  naturally  immune  to  anthrax.     The  insusceptibility 
of  this  species  has  however  been  overcome  experimentally  in  several  ways. 
Pasteur,  for  instance,  succeeded  in  rendering  fowls  susceptible  by  keeping 
their  legs  in  cold  water  at  a  temperature  of  25°  C.  :    Wagner  obtained  the 
same  result  by  lowering  the  temperature  by  means  of  repeated  inoculations 
of  antipyrin  :    Canalis  and  Morpugo  by  experimenting  while  the  animals 
were  fasting,  etc. 

The  pigeon  is  less  highly  immune  than  the  fowl  and  readily  succumbs 
to  the  inoculation  of  anthrax  bacilli  into  the  anterior  chamber  of  the  eye. 
Young  pigeons  are  much  more  susceptible  than  adults. 

A  virus  which  has  been  passed  through  dogs  readily  kills  pigeons  (Martel).  The 
virulence  of  the  bacillus  is  also  increased  by  passage  through  pigeons  and  after 
several  such  passages  a  virus  is  obtained  which  on  sub-cutaneous  or  intra- muscular 
inoculation  will  kill  a  full-grown  pigeon  and  even  a  fowl  (MetchnikofF). 

8.  Cold-blooded  vertebrata. — Batrachians  are  immune  to  anthrax.     Gibier 
has,  however,  been  able  to  infect  frogs  by  keeping  the  inoculated  animals 
in  water  at  35°  C.     Catterina  succeeded  in  infecting  newts. 

Sabrazes  and  Colombot  have  shown  that  hippocampi  (Lophobranchiata)  are 
susceptible  to  anthrax.  These  animals  kept  under  the  normal  conditions  of  their 
existence  die  a  few  days  after  the  sub-cutaneous  inoculation  of  0*25  c.c.  of  a  broth 
culture.  Sabrazes  and  Colombot  attribute  this  susceptibility  to  the  absence  of  a 
spleen  and  the  small  number  of  leucocytes  in  the  blood  of  hippocampi. 

9.  Invertebrata. — Slugs  are  naturally  immune  to  anthrax  (Kowalewsky), 
but  Lode  has  succeeded  in  infecting  them  by  keeping  them  at  a  temperature 
of  32°  C.  and  inoculating  them  in  the  body  cavity. 

2.  Methods  of  inoculation. 

1.  Sub-cutaneous  inoculation. — Inject  sub-cutaneously  with  the  ordinary 
precautions  a  few  drops  of  anthrax  blood,  or,  better  a  few  drops  of  a  young 
broth  culture  of  anthrax  bacilli. 

Broth  cultures  incubated  at  37°  C.  for  2  or  3  days  are  more  virulent  than  the 
blood  with  which  they  were  sown,  probably  on  account  of  the  presence  of  antibodies 
in  the  anthrax-infected  blood. 

2.  Ingestion. — -Pasteur  and  Chamberland  infected  sheep  with  anthrax  by 
mixing  thorns  and  splinters  of  wood  previously  watered  with  spore-bearing 
cultures  of  anthrax  with  their  food. 

3.  Intra- venous  inoculation. — Inject  a  small  quantity  'of  a  broth  culture 
into  a  vein.     Anthrax  blood  should  never  be  used  for  intra-venous  inoculation 
for  fear  of  producing  a  fatal  embolism. 

4.  Intra-muscular  inoculation. — This  method  is  sometimes  used  in  the  case 
of  birds,  the  ordinary  technique  being  adopted. 

3.  Symptoms  and  lesions  in  experimental  animals. 

The  symptoms  and  lesions  in  the  guinea-pig  and  rabbit  after  sub-cutaneous 
inoculation  will  be  described,  as  these  are  the  animals  most  commonly  used 
for  experimental  purposes,  and  this  the  usual  form  of  infection. 

A.  Symptoms.  Local  reaction. — Eight  to  fifteen  hours  after  inoculation 
a  small  oedematous  puffiness  appears  around  the  site  of  inoculation.  The 
neighbouring  glands  then  become  swollen  and  the  temperature  rises  1°  or 
2°C. 

General  reaction. — For  the  first  24  or  30  hours  in  the  case  of  the  guinea- 
pig  and  for  30-50  hours  in  the  case  of  the  rabbit  the  animal  shows  no  symptoms, 


520  THE   ANTHRAX   BACILLUS 

but  later  it  becomes  restless,  the  respiration  is  accelerated,  and  there  is  fre- 
quency of  micturition ;  the  animal  then  huddles  itself  up  and  becomes  drowsy, 
the  temperature  falls  to  below  normal  (34°  or  30°  C.),  coma  supervenes  and 
the  animal  dies  in  a  few  minutes. 

B.  Lesions.  1.  Local  lesion. — At  the  site  of  inoculation  there  is  more  or 
less  oadema  of  the  sub-cutaneous  cellular  tissue,  the  exudate  is  gelatinous, 
somewhat  red,  very  poor  in  leucocytes  and  contains  numerous  organisms 
(gelatinous  ffidema).  The  neighbouring  lymphatic  glands  are  enlarged, 
ecchymosed,  surrounded  by  an  oedematous  area  and  contain  numerous 
bacilli. 

2.  The  blood. — The  bacilli  appear  in  the  blood  about  15  hours  after  inocula- 
tion, and  at  the  time  of  death  the  blood  is  literally  swarming  with  them. 
The  blood  is  very  dark  in  colour  and  of  the  consistence  of  pitch  ;  it  coagulates 
slowly  and  does  not  become  red  on  exposure  to  the  air.     There  is  an  hyper- 
leu  cocytosis,  the  red  cells  are  deformed  and  in  film  preparations  agglutinate 
in  irregular  masses.     The  veins  are  congested. 

3.  Internal  organs. — The  anthrax  bacillus  is  a  strict  ae'robe,  and  the  ana- 
tomical characteristic  of  the  disease  is  the  presence  of  the  bacilli  in  the  blood 
capillaries.     The  parenchyma  of  the  internal  organs  contains  no  bacilli  unless 
the  latter  have  reached  there  through  rupture  of  the  blood  vessels.     The 
glandular  and  epithelial  cells  are  always  found  practically  undamaged  and, 
contrary  to  what  occurs  in  other  infections,   lesions  of  degeneration  are 
never  seen. 

The  spleen  is  swollen  and  diffluent :  it  contains  a  veritable  felting  of  bacilli. 

Liver,  lungs  and  glands. — The  blood  capillaries  are  engorged  with  bacilli 
while  the  epithelial  cells  are  intact.  In  the  bile  a  very  few  bacilli  are  some- 
times found  which  have  escaped  from  a  ruptured  blood  vessel.  In  females 
during  the  period  of  lactation  bacilli  may  pass  into  the  milk  in  a  similar 
manner  (Straus  and  Chamberland). 

Kidney. — The  glomerular  and  inter-tubular  capillaries  are  engorged  with 
bacilli :  the  epithelium  is  intact.  Rupture  of  small  blood  vessels  often  takes 
place  and  bacilli  then  pass  into  the  tubules  and  so  into  the  urine  (Chamber- 
land  and  Straus). 

Mesentery  and  intestine. — The  vessels  of  the  mesentery  and  of  the  intestinal 
villi  are  engorged  with  bacilli. 

Anthrax  bacilli  are  present  in  the  excreta  of  infected  animals.  Cinsa  and  Fenea 
have  shown  that  the  numbers  of  bacilli  increase  with  the  duration  of  the 
disease.  In  the  intestine  as  in  the  soil  the  conditions  are  favourable  for  spore 
formation. 

Muscles.  Nervous  system. — Very  few  bacilli  are  found  in  the  muscular 
and  nervous  tissues,  etc. 

Placenta. — In  pregnant  females  the  bacilli  do  not  pass  through  the  placenta 
so  long  as  the  vessels  are  intact ;  but  the  vessels  frequently  rupture,  and  so 
the  bacilli  are  enabled  to  pass  the  placental  filter  and  infect  the  foetus  (Straus 
and  Chamberland,  Perro^ito,  and  Toussaint). 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearances  and  staining  reactions. 

Under  the  microscope  the  bacillus  presents  three  different  appearances.  In 
the  tissues  of  man  and  animals  infected  with  anthrax  it  occurs  exclusively 
in  the  bacillary  form  ;  in  cultures,  filamentous  forms  and  spores  are  found 
in  additiori. 


MORPHOLOGY  521 

(i)  The  bacillary  form. 

In  the  blood  of  animals  infected  with  anthrax  the  bacilli  occur  as  small 
rods  5-10/A  by  1-1 '5/x,  straight,  flexible,  non-motile  and  staining  uniformly. 


FIG.   249. — Bacillus  anthracis.     Blood  film  from  an  ox  dead  of  anthrax. 
Gram's  stain.     (Oc.  2,  obj.  ^th,  Zeiss.) 

The  organisms  are  arranged  singly  or  in  short  chains  of  two  or  three  bacilli : 
sometimes  the  individuals  in  the  chains  are  separated  from  one  another  by 
such  a  small  space  that  they  appear  rather  as  one  long  filamentous  bacillus. 


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FIG.  250. — Bacillus  anthracis.     Section  of  liver.     Gram's  method  and  eosin. 
(Oc.  -2,  obj.  y^th,  Zeiss.) 

If  unstained  preparations  be  examined  the  bacilli  appear  transparent 
like  glass. 

In  the  gelatinous  oedema  the  bacilli  are  a  little  longer  than  in  blood  ;  it 
is  also  to  be  noted  that  the  length  of  the  bacilli  varies  in  different  animals 
and  also  according  to  the  virulence  of  cultures.  A  slightly  attenuated  virus 
gives  long  forms,  more  virulent  organisms  are  shorter  and  thick-set. 


522  THE   ANTHRAX   BACILLUS 

In  the  living  tissues  the  bacilli  never  form  spores,  reproduction  taking 
place  solely  by.  fission. 

Staining  methods.  —  The  bacillus  is  readily  stained  by  all  the  basic  aniline 
dyes.  It  is  gram-positive.  After  staining  it  will  be  noticed  that  the  ends 
are  never  rounded  but  always  square  cut.  Under  very  high  magnification 
the  ends  show  an  outline  which  is  [ragged  or]  sinuous  rather  than  straight, 
as  though  the  bacillus  had  been  roughly  broken.  This  appearance  is  charac- 
teristic of  the  organism.  On  account  of  its  large  size  an  oil-immersion  lens 
is  not  generally  necessary  for  the  examination  of  microscopical  preparations, 
a  high  -power  dry  objective  being  of  sufficient  magnifying  power. 

Staining  of  films  and  sections.  —  Gram's  method  will  be  used  for  choice  in 
searching  for  and  identifying  the  organism  in  blood  films,  smears  and  sections. 
The  mesentery  and  films  and  smears  of  the  internal  organs  should  be  stained 
by  the  double  method  (eosin  and  violet),  sections  by  the  double  or  triple 
method  (Orth's  picro-carmine  and  violet).  The  technique  is  described 
fully  at  p.  219. 

Capsules.  —  The  bacillus  of  anthrax  in  the  blood  and  in  smears  from  the 
spleen  frequently  shows  a  very  distinct  capsule  (Serafini).  This  capsule  is 


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A 


FlG.  251. — Bacillus  anthracis.     Blood  film   from  an  ox   dead  of  anthrax. 
Jenner's  stain.    (Oc.  2,  obj.  ^th,  Zeiss.) 

visible  in  preparations  stained  by  carbol-thionin  or  carbol-blue  and  may 
also  be  demonstrated  by  the  ordinary  methods  for  staining  capsules  (p.  148). 
[In  examining  blood  from  an  animal  suspected  to  have  died  from  anthrax 
the  film  is  best  stained  either  with  Jenner's  stain  or  with  an  alkaline  solution 
of  methylene  blue.  With  Jenner's  stain  the  organism  shows  a  well  marked 
capsule.  The  bacillus  itself  stains  blue  while  the  capsule  assumes  a  delicate 
pink  tint.  This  method  has  been  found  of  the  utmost  value  in  practice  and 
enables  a  distinction  to  be  drawn  at  sight  between  the  anthrax  bacillus  and 
the  bacillus  of  malignant  oedema,  an  organism  by  no  means  infrequently 
found  in  blood-films  made  from  dead  animals.] 

(ii)  Filamentous  form. 

In  cultures  the  anthrax  bacillus  usually  occurs  in  long  filaments  which  are 
best  studied  in  a  broth  culture  in  which  they  are  longer  than  on  solid  media. 


MORPHOLOGY 


523 


FIG.  252.— Bacillus  anthracis.     Broth 
Iture. 
Reich.) 


The  bacterial  filaments  are  1-2/x  broad,  very  long,  wavy,  cylindrical, 
flexible  and  fitting  one  within  the  other  like  the  strands  in  a  skein  of  silk.  They 
are  squarely  cut  at  the  end,  are  never  branched 
and  appear  to  be  absolutely  non-motile. 

R.  Dupond  has  however  noticed  a  very  slow 
and  flexuous  movement  in  a  broth  medium  con- 
timing  no  salt  when  examined  on  a  warm  stage  at 
38°  C.  After  making  several  sub-cultures  of  the 
first  vaccine  of  the  Institut  Pasteur  on  glycerin- 
sagar-agar  he  was  able  by  staining  to  demonstrate 
flagella  arranged  regularly  around  the  bacilli. 

Staining  methods. — Like  the  bacillary  form, 
the  filaments  stain  with  the  basic  aniline 
dyes  (carbol-thionin  is  best)  and  are  gram- 
positive. 

When  stained  the  filaments  are  seen  to  be  ?ul^re-  c^boi-thionin.  (Oc.ii.obj.  8, 
made  up  of  an  hyaline  sheath  enclosing  a 
number  of  homogeneous  protoplasmic  segments  separated  one  from  another 
by  transverse  partitions,  each  segment  representing  a  cell  which  rapidly 
forms  a  spore. 

(iii)  Spores. 

To  study  the  formation  and  development  of  spores  it  is  necessary  to 
examine  a  hanging-drop  preparation.  The  best  method  is  to  use  a  Koch's 
cell ;  place  a  drop  of  aqueous  humour  on  the  cover-glass  and  sow  it  with  a 
trace  of  anthrax  blood. 

If  the  cell  be  kept  at  a  temperature  of  35°-37°  C.  a  small  refractile  point  will 

appear  within  the  protoplasm  of  the  bacilli 
in  the  course  of  a  few  hours.  This  point 
increases  in  size  and  by  reason  of  its  re- 
fractibility  becomes  a  distinctly  visible  ovoid 
body.  The  spore  is  the  resistant  form  of 
the  bacillus. 

Spore  formation  only  takes  place  under  cer- 
tain conditions  :  the  presence  of  free  oxygen 
is  essential  and  the  cultures  must  be  grown  at 
a  temperature  between  18°  and  41*5°  C.  Above 
42°  C.  spores  are  not  formed. 

The  protoplasm  of  the  original  bacillus 
soon  breaks  up  leaving  the  spore  surrounded 
merely  by  the  delicate  membrane  which 
enclosed  the  organism  in  its  filamentous 

su. -uu^c/w/w-o      wrvuii  w/i>to.        ajAVAiici.   Q/»  1       j_l     *          *  "1  "  1" 

method.  TO  demonstrate  spores,  x  925.  iorm  and  this  in  turn  disappears  leaving 
SS^UrtiS>  EssentMs  of  Practical  Bac~  the  spore  free.  No  one  bacillus  ever  gives 

rise  to  more  than  one  spore  and  this  is 

always  smaller  than  the  mother  cell ;  some  of  the  bacilli  are  sterile  so  that 
a  spore  does  not  form  in  every  cell  of  the  chain. 

If  the  medium  is  sufficiently  nutritive,  the  spore  will  soon  begin  to  change 
into  a  bacillus.  It  first  increases  in  size  and  loses  its  refractibility,  then  the 
enveloping  membrane  is  absorbed  leaving  the  protoplasm  free,  finally  it 
elongates  and  assumes  the  bacillary  form  (De  Bary). 

Staining  methods. — The  spores  of  anthrax  may  be  stained  by  the  methods 
described  in  Chap.  IX.  It  is  difficult  to  stain  the  spores  by  the  double 
method,  but  if  attempted  absolute  alcohol  should  be  used  for  decolonization 
and  not  acids  (p.  147). 


FIG.   253.— Bacillus  anthracis.     Holler's 


524 


THE  ANTHRAX  BACILLUS 


(iv)  Involution  forms. 

In  non-sporing  cultures  obtained  by  methods  which  will  be  described  later, 
involution  forms  are  frequently  seen  :  these  are  curved  and  the  ends  more 
or  less  swollen  (fig.  254).  In  attenuated  cultures  Chauveau  described  abnormal 

forms,  sometimes  short  and  sometimes  thin 
and  filiform,  with  a  spore  at  the  end  giving 
the  bacillus  the  appearance  of  a  nail. 

In  collodion  sac  cultures  in  dogs  the  anthrax 
bacillus  loses  its  virulence  and  occurs  as  single 
coccal-like  bacilli  (Phisalix)  which  stain  by  Gram's 
method  and  liquefy  gelatin.  These  forms  seem  to 
have  a  certain  degree  of  stability  and  constitute 
a  true  variety  (B.  anthracis  brevigemmans  of 
Phisalix). 


V 

/ 


y 


2.  Cultural  characteristics. 


FIG.  zzi—  Bacillus  anthracis. 
luarn 


invo- 
°D 


Conditions  of  growth.  —  The  bacillus  is  essen- 

an  ae'robic  organism. 

Rosenthal  has  shown  that  the  anthrax  bacillus 
can  be  converted  into  a  facultative  anaerobe  by  growing  it  in  media  containing  smaller 
and  smaller  quantities  of  oxygen.  Having  changed  into  an  anaerobe  it  no  longer 
forms  spores  and  is  much  less  resistant  to  adverse  conditions. 

[Parry  Laws  several  years  ago  showed  that  when  the  anthrax  bacillus  was  sown  in 
broth  and  incubated  in  a  complete  vacuum  the  organism  when  examined  microscopi- 
cally showed  no  spores,  exhibited  a  peculiar  morphology  and  died  in  ten  to  twenty  days 
but  if  sub-cultivated  in  broth  and  grown  under 
aerobic  conditions  the  organism  reassumed  its 
normal  characteristics.  ] 

The  optimum  temperature  is  35°  C.  but 
growth  occurs  at  any  temperature  between 
14°  C.  and  43°  C.  and  spores  form  when  the 
cultures  are  incubated  at  temperatures  be- 
tween 18°  C.  and  42°  C.  The  bacillus  re- 
quires a  neutral  or  slightly  alkaline  medium. 

Characters  of  growth.  Broth.  —  After  in- 
cubation for  a  few  hours  at  35°  C.  delicate 
flakes  make  their  appearance  in  the  medium 
which  subsequently  increase  in  size  and 
adhering  together  fall  to  the  bottom  of  the 
tube,  leaving  the  medium  clear. 

Gelatin.  —  The  anthrax  bacillus  liquefies 
gelatin. 

(a)  Stab  cultures.  —  After  incubating  for  48 
hours  at  20°  C.  a  whitish  track  appears  along 
the  line  of  sowing  and  from  this  numerous 
delicate,  downy  filaments  soon  grow  out  at 
right  angles  giving  an  appearance  similar 
to  the  "  tree  of  Saturn  "  (fig.  255).  The 
growth  continues  to  increase  in  amount  and 
the  lateral  offshoots  become  thicker  :  later 
the  gelatin  begins  to  liquefy  at  the  upper  part  of  the  tube  and  the  liquefaction 
gradually  extends  through  the  medium,  until  in  10  or  12  days  the  whole  is 
liquefied.  At  this  stage  the  growth  has  the  appearance  of  large  white  flakes 
floating  in  a  clear  liquid  ;  finally  the  flakes  fall  to  the  bottom  of  the  tube. 


FIGS.  255, 256. — Bacillus  anthracis.    Stab 
cultures  on  gelatin  at  20°  C.  (3  days  and 


MORPHOLOGY 


525 


Sometimes  the  arborization  is  wanting  in  which  case  the  culture  is  reduced  to  the 
central  streak.  Branching  cultures  are  especially  observed  when  the  medium  is 
sown  with  anthrax  blood. 

(b)  Plate  cultures. — Towards  the  second  day  small  greyish-white  points 
appear  scattered  through  the  gelatin  on 
the  plates. 

These  points  increase  rapidly  in  size 
and  form  brownish,  granular  rounded 
spots  with  wavy  margins.  A  low  power 
lens  shows  these  colonies  to  be  made  up 
of  interlaced  filaments  which  give  them 
the  appearance  of  a  tangled  ball  of  silk. 
Towards  the  fourth  or  fifth  day  the 
colonies  look  as  though  they  were  made 
up  of  twisted  threads  and  resemble  curly 
hair  [Judge's  wig  appearance] .  The  gela- 
tin then  becomes  liquefied  around  the 
colonies  which  break  up  and  form  flakes 
floating  in  the  liquefied  gelatin. 

Agar. — After  incubation  for  24  hours 
at  35°-37°  C.  a  whitish  streak  appears  on 
the    sloped   surface   of   the    agar  which 
rapidly  thickens,  becomes  rather  dry  and  friable  and  has  lightly  notched 
borders.     The  growth  on  agar  is  not  very  characteristic. 

Potato. — On  this  medium  at  a  temperature  of  35°-37°  C.  there  appears 
after  the  second  day  a  whitish  deposit  which  rapidly  thickens  and  assumes  a 

dull  white  colour,  becoming  brown  on  keeping 
(fig.  258). 

Serum.  Liquid  serum. — After  incubation  for 
2  days  at  35°  or  37°  C.  flakes  are  seen  floating 
in  the  medium  which  subsequently  fall  to  the 
bottom  of  the  tube. 

Coagulated  serum. — The  growth  appears  as  a 
dull  white  streak  becoming  greyish  after  a  few 
days  ;  the  serum  is  partly  liquefied. 

Milk. — When  sown  in  a  tube  of  milk  and  incu- 
bated at  35°  or  37°  C.  coagulation  takes  place 

towards  the  third  or  fourth  day.  The  coagulum  is  redissolved  about  the 
end  of  the  week.  If  a  flask  be  used  no  coagulation  takes  place  but  the  milk 
acquires  a  yellowish  colour. 

Litmus-lactose-gelatin. — The  medium  is  slightly  reddened  by  the  growth 
of  the  anthrax  bacillus. 


x  240  x  530 

FIG.  257. — Bacillus  anthracis.  Impression 
preparation  from  glycerin-agar.  From  Cur- 
tis' Essentials  of  Practical  Bacteriology. 


YlG.ZM.-Bactilusanthracis.  Culture 

on  potato  (3  days  at  37°  c.). 


SECTION   III.— BIOLOGICAL  PROPERTIES. 
1.  Viability.    Resistance. 

The  non-sporing  bacillus  is  quickly  killed  by  exposure  to  a  temperature 
above  50°  C.  At  51°  C.  anthrax  blood  is  sterilized  in  half  an  hour.  Exposure 
even  to  very  low  temperatures  (  -  100°  C.  for  1  hour)  does  not  kill  the  bacillus. 
The  absence  of  oxygen  or  immersion  in  compressed  oxygen  kills  the  non- 
sporing  bacillus. 

Spores,  the  resistant  form  of  the  organism,  possess  the  full  virulence  of  the 
bacillus.  In  soil  the  spores  can  retain  their  vitality  as  well  as  their  virulence 
for  more  than  15  years  (Sirena).  Spores  in  the  moist  condition  resist  a  tern- 


526  THE   ANTHRAX   BACILLUS 

perature  of  70°  C.  for  a  very  long  time  and  a  temperature  of  85°  C.  for  5 
minutes.  In  the  dried  state  and  especially  in  presence  of  albuminous  matter 
such  as  blood  their  resistance  is  even  greater ;  they  will  then  tolerate  a  tem- 
perature above  100°  C.  and  resist  the  action  of  absolute  alcohol,  of  compressed 
oxygen,  complete  absence  of  oxygen,  exposure  to  sunlight,  etc. 

Arloing  however  has  shown  that  in  cultures  spores  resist  the  action  of  sunlight 
less  well  than  the  non- spore- bearing  bacillus.  This  may  be  explained  by  assuming 
that  sunlight  acts  more  powerfully  on  young  bacilli  developing  from  spores  than  on 
the  full-grown  bacillus. 

The  spore  only  develops  into  a  bacillus  in  presence  of  free  oxygen. 

The  non-sporing  bacillus. — Pasteur  noted  that  in  old  cultures  on  gelatin  the 
organism  sometimes  loses  its  power  of  forming  spores. 

If  broth  be  sown  with  anthrax  blood  and  incubated  at  42 '5°  C.  the  bacillus 
grows  but  no  spores  are  formed  :  in  the  rods  small  bright  granules  are  seen — 
the  false  spores  of  Chauveau — but  these  have  none  of  the  properties  charac- 
teristic of  spores. 

Non-sporing  bacilli  obtained  in  this  way  when  resown  in  a  medium  kept 
at  the  optimum  temperature  soon  revert  to  the  sporing  form.  To  obtain  a 
definitely  non-sporing  variety  one  of  the  following  methods  must  be  adopted  : 

A.  The  carbolic  acid  method  of  Roux.  Recommended. — 1.  Distribute 
about  10  c.c.  of  a  slightly  alkaline  peptonized  veal  broth  into  each  of  a 
number  (30  to  50)  of  tubes. 

2.  Divide  these  tubes  into  series  of  ten  each.     Number  the  tubes  of  each 
series  1,  2,  3,  .'.  .  10,  and  to  each  tube  add  a  1  per  cent,  aqueous  solution 
of  carbolic  acid  (without  alcohol)  in  the  following  quantities  : 

to  tube  No.  1  equivalent  to  10  *00  of  carbolic  acid  =  0'2  c.c.  of  the  carbolic  acid  solution. 
<>     No.  2  „       Iirf0o  „  =0-4  c.c. 

-,     No.  3  „       I¥|Fo  „  =0'6  c.c. 

»     No.  10  „        ^^U  „  =2-0  c.c. 

3.  Plug  the  tubes  with  wool,  seal  off  the  ends  in  the  blow-pipe  above  the 
plug  to  prevent  evaporation  of  the  carbolic  acid,  and  then  sterilize  in  the 
autoclave. 

4.  When  cool  open  the  tubes  and  sow  with  a  drop  of  anthrax  blood  :   care 
should  be  taken  to  drop  the  blood  into  the  broth  and  not  on  to  the  side  of 
the  tube. 

5.  Cover  the  mouths  of  the  tubes  with  india-rubber  caps  and  incubate 
at  33°-37°  C. 

6.  After  incubating  for  about  10  days  examine  the  cultures.     In  some 
of  the  tubes,  those  containing  the  largest  amount  of  carbolic  acid  (tubes 
7-10,  for  instance)  growth  will  have  been  altogether  inhibited  ;    the  tubes 
numbered  say  1  to  3  will  contain  both  bacilli  and  spores  ;  the  tubes  numbered 
3,  4,  5,  6  will  contain  bacilli  but  no  spores. 

To  determine  whether  or  not  spores  are  present  draw  up  a  little  of  the  culture 
into  a  very  fine  Pasteur  pipette  constricted  below  the  wool  plug  (p.  75).  Seal  the 
ends  of  the  pipette  in  the  flame  and  put  it  in  a  water  bath  at  65°  C.  or  70°  C.  for 
15  minutes  or  so.  If  the  bacillus  is  non-spore-bearing  it  is  killed  at  this  temperature 
so  that  when  the  heated  contents  of  the  pipette  are  sown  in  broth  the  medium 
remains  sterile.  If  on  the  other  hand  the  bacteria  are  spore-bearing  growth  takes 
place. 

Sometimes  none  of  the  tubes  contain  asporogenic  bacteria,  and  at  other 
times  a  large  number  are  obtained  which  however  may  revert  to  the  sporing 
form  after  a  few  sub-cultures  in  broth  at  33°-37°  C. 

Surmont  and  Arnold  have  shown  that  the  faculty  of  becoming  asporogenic  varies 
much  among  bacteria  recovered  from  different  sources.  A  bacterium  obtained 


BIOLOGICAL   PROPERTIES  527 

from  the  Institut  Pasteur  easily  became  asporogenic  in  the  hands  of  these  investi- 
gators although  they  failed  when  they  worked  with  a  bacterium  isolated  by  themselves 
from  a  case  of  human  anthrax. 

B.  The  bichromate  method  of  Chamberland  and  Roux.— Bichromate  of 
potassium  is  used  in  the  same  way  as  the  carbolic  acid  in  the  previous  experi- 
ment but  in  different  proportions.     A  solution  of  1  in  2000  in  broth  is  the 
best. 

C.  Other  methods. — The  following  may  be  placed  on  record  here  but  none 
of  them  have  up  till  the  present  given  satisfactory  results.     The  method  of 
von  Behring  depends  upon  the  Use  of  rosolic  and  hydrochloric  acids  ;    that 
of  Phisalix  is  based  upon  the  application  of  heat  (successive  cultures  at 
42'5°C.).     More  recently  Phisalix  and  also  Bormans  have  advised  repeated 
sub-cultivation  on  horse  or  dog  serum  ;    Phisalix  also  recommends  collodion 
sac  cultures  in  the  peritoneal  cavities  of  dogs  and  other  animals. 

2.  Virulence.    Attenuation.    Pasteur's  vaccination. 

Virulence. — In  man  one  attack  of  anthrax  confers  an  absolute  immunity. 
As  a  rule,  anthrax  is  fatal  to  domestic  animals,  but  Pasteur  found  that  cows 
which  had  recovered  from  an  attack  of  the  disease  were  able  to  resist  without 
ill-effect  the  subsequent  inoculation  of  a  very  virulent  organism.  It  had  been 
observed  also  that  in  the  district  of  La  Beauce  some  of  the  sheep  were  immune 
to  anthrax,  and  by  way  of  explanation  the  suggestion  had  been  made  that 
these  immune  animals  had  already  suffered  from  an  abortive  attack  of  the 
disease.  From  these  facts  Pasteur  inferred  that  if  he  could  infect  animals 
with  a  mild  form  of  anthrax,  he  would  be  able  to  render  them  immune  to 
the  epizootic  disease. 

Attenuation. — -To  demonstrate  the  truth  of  this  inference  an  attenuated 
virus  had  to  be  prepared.  But  since  the  virulence  of  the  bacterium  is  retained 
intact  and  perpetuated  by  the  spore  which  originates  in  it  the  bacillus  had  to 
be  prevented  from  forming  spores.  The  method  by  which  this  was  and  is 
still  done  is  as  follows  : 

1.  Sow  a  flask  of  broth  with  anthrax  blood  and  incubate  at  42*5°  C.     The 
organism  grows  but  does  jaot  form  spores. 

The  virulence  of  the  bacillus  is  at  first  considerable  but  soon  diminishes 
and  after  about  8-10  days  is  harmless  on  inoculation  to  guinea-pigs  and 
rabbits.  This  attenuation  is  due  to  the  combined  action  of  air  and  heat 
on  the  micro-organism. 

If  the  attenuated  culture  be  inoculated  into  a  sheep  the  animal  suffers 
from  a  very  mild  attack  of  anthrax  and,  after  it  has  recovered,  it  will  be  found 
to  be  capable  of  resisting  the  inoculation  of  a  fully  virulent  organism. 

That  is  to  say,  the  inoculation  of  an  attenuated  virus  confers  an  immunity 
upon  the  animal  inoculated. 

2.  If  the  attenuated  bacillus  be  sown  in  a  flask  and  incubated  at  33°-37°  C. 
it  will  again  form  spores  but  the  virulence  will  be  that  of  the  bacillus  in  which 
they  develop  ;    in  this  way  the  virulence  is  fixed  and  may  be  indefinitely 
perpetuated. 

Restitution  of  the  virulence. — The  virulence  of  an  attenuated  bacillus  which  has 
become  saprophytic  can  be  restored  by  passage  through  suitable  animals. 

For  instance,  take  a  bacillus  which  fails  to  kill  an  adult  mouse,  and  inoculate  it 
into  a  mouse  which  has  just  been  born.  The  latter  dies  in  2  or  3  days.  Inoculate 
a  little  of  the  blood  of  this  first  animal  into  a  second  mouse  3  days  old  and  after  it 
is  dead  inoculate  some  of  its  blood  into  a  mouse  6  days  old.  Inoculate  the  blood  of 
the  latter  into  an  adult  mouse  then  some  of  the  blood  of  the  adult  mouse  into  a 
young  guinea-pig  and  so  on  through  an  adult  guinea-pig,  a  rabbit,  a  sheep  and  a  bovine 
animal  :  finally  a  fully- virulent  organism  will  be  recovered. 


528  THE   ANTHRAX   BACILLUS 

Pasteur's  method  of  vaccination. — The  more  severe  the  vaccinating  infection 
the  more  highly  immunized  will  the  animal  be. 

On  the  other  hand  there  is  the  danger  that  if  a  powerful  vaccine  be  inocu- 
lated in  the  first  instance  the  animal  may  die  and  the  experiment  have  to 
be  recommenced. 

In  practice  a  compromise  is  effected  by  using  two  vaccines.  The  first 
inoculation  is  made  with  a  very  weak  vaccine  which  will  kill  mice  but  has 
no  ill-effect  on  rabbits  and  sheep  (premier  vaccin).  The  second  inoculation 
is  given  12  days  later  :  it  is  somewhat  more  virulent  than  the  first  vaccine 
and  will  kill  mice  and  guinea-pigs  and,  twice  out  of  six  or  eight  times, 
rabbits  (second  vaccin).  Immunity  is  established  12  days  after  the  second 
inoculation. 

The  vaccine  prepared  by  the  Institut  Pasteur  is  supplied  to  veterinary 
surgeons  at  a  trifling  cost  in  tubes  containing  100  doses.  The  first  and 
second  vaccines  are  inoculated  successively,  the  dose  for  a  cow  being  0'25  c.c. 
and  for  a  sheep  0'125  c.c. 

As  a  rule,  the  inoculations  are  given  as  follows  :  The  first  vaccine  is  inocu- 
lated into  the  internal  surface  of  the  right  thigh  and  the  second  into  the 
internal  surface  of  the  left  thigh,  an  interval  of  12  days  elapsing  between 
the  two  operations.  The  vaccine  must  be  used  as  soon  as  it  is  procured  and 
the  inoculations  must  be  done  with  a  sterile  syringe.  It  is  important  not  to 
use  a  contaminated  vaccine,  because  by  contamination  it  has  lost  its  properties. 

Immunization  of  small  animals. — The  immunization  of  small  animals  is 
difficult  but  necessary  for  laboratory  investigations.  Rabbits  and  guinea- 
pigs  are  so  highly  susceptible  that  death  often  occurs  during  the  process. 
For  such  animals  three  vaccines  of  different  virulence  ought  to  be  available, 
the  first  very  attenuated,  the  second  the  premier  vaccin  and  the  third  the 
second  vaccin  described  above.  The  inoculations  should  be  carried  out  very 
cautiously. 

Marchoux  succeeded  in  immunizing  laboratory  animals  by  using  only  the 
"  sheep  vaccines."  These  vaccines  were  grown  at  37°  C.  in  a  peptonized 
veal  broth  and  used  when  24  hours  old.  The  first  vaccine  given  under  the 
skin  of  a  rabbit  consisted  of  0'5  c.c.  (maximum  non-fatal  dose)  of  the  more 
attenuated  of  the  two  sheep  vaccines.  It  caused  a  rise  of  temperature, 
diarrhoea  and  loss  of  weight.  The  second  vaccine  given  12  days  later  con- 
sisted of  a  double  dose  of  the  same  vaccine.  The  third  inoculation  given 
after  another  interval  of  12  days  consisted  of  0*25  c.c.  of  the  more  virulent  of 
the  sheep  vaccines  (second  vaccin)  and  12  days  later  again  a  fourth  inoculation 
of  0'5  c.c.  of  the  latter  vaccine  was  inoculated.  A  week  after  the  last  inocula- 
tion the  animal  was  tested  by  the  inoculation  of  a  few  drops  of  anthrax  blood 
under  the  skin.  If  the  reaction  was  not  too  violent  the  immunizing  process 
was  continued  and  completed  by  the  frequent  inoculation  of  anthrax  blood 
or  of  virulent  cultures  24  hours  old.  Marchoux  succeeded  in  immunizing 
some  of  his  rabbits  to  such  a  degree  that  they  were  able  to  resist  the  daily 
inoculation  of  1  c.c.  of  fully  virulent  bacilli  while  other  rabbits  could  resist 
the  inoculation  every  fifth  day  of  gradually  increasing  doses  up  to  20  c.c. 

Immunization  by  avirulent  cultures. — De  Christmas  showed  that  white  rats  can 
be  immunized  with  totally  avirulent  cultures.  He  injected  into  the  peritoneal 
cavity  of  white  rats  1  c.c.  of  a  watenr  emulsion  of  a  young  avirulent  culture  on 
three  different  occasions  at  intervals^  of  a  month.  The  material  for  sowing  the 
cultures  was  taken  from  a  non-sporing  strain  which  had  been  grown  for  a  number 
of  years  at  the  Institut  Pasteur  and  which  was  totally  devoid  of  virulence  for  white 
rats.  All  the  animals  vaccinated  in  this  manner  were  inoculated  a  month  after 
the  last  immunizing  inoculation  with  a  large  dose  of  a  virulent  anthrax  bacillus 
which  was  certainly  fatal  to  control  animals,  and  were  found  to  be  immune. 


TOXIN  529 

3.  The  toxin  of  anthrax. 

The  nature  of  the  toxin  secreted  by  the  anthrax  bacillus  is  not  yet  definitely 
determined. 

The  toxalbumin  which  Hankin  prepared  from  cultures  of  the  bacillus  grown  in 
Liebig's  broth  containing  fibrin  and  that  which  Brieger  and  Frsenkel  extracted  from 
carcases  dead  of  anthrax  were  only  obtained  by  complicated  processes  hardly  com- 
patible with  the  isolation  of  a  very  delicate  chemical  substance.  It  is  to  be  feared 
that  impurities  were  responsible  for  many  of  the  results  which  followed  the  inocula- 
tion of  these  toxins. 

Sidney  Martin  obtained  an  albumose  in  solutions  of  alkali  albumin  which  in  doses 
of  3  eg.  killed  mice  weighing  22  grams  with  symptoms  very  similar  to  those  seen 
in  anthrax  septicaemia. 

Marmier's  toxin. — Marmier  obtained  an  active  toxin  by  growing  the  organism 
at  a  low  temperature  in  a  solution  of  pure  peptone  containing  glycerin. 

Marmier's  medium. — To  prepare  Marmier's  medium  the  first  step  is  to  purify 
ordinary  commercial  peptone,  thus  :  dissolve  a  certain  amount  of  peptone  in  water;, 
add  sufficient  sulphate  of  ammonium  to  saturate  the  solution  at  100°  C.  then  boil 
for  a  few  minutes  and  filter.  To  the  filtrate  add  sufficient  barium  hydroxide  to 
precipitate  all  the  sulphuric  acid  present.  Heat  the  mixture  for  several  hours  to 
a  temperature  near  the  boiling  point  to  drive  off  the  ammonia :  filter  to  remove 
the  barium  sulphate  and  keep  the  filtrate  boiling  while  passing  a  current  of  air 
through  it  to  remove  all  traces  of  ammonia  ;  then  pass  through  it  a  current  of  carbon- 
dioxide  to  precipitate  the  excess  of  barium  hydroxide,  and  finally  filter.  With 
this  purified  peptone  solution  the  following  medium  is  prepared : 

Water, ...       1000      c.c. 


Peptone, 
Common  salt, 
Sodium  phosphate, 
Potassium  phosphate, 
Pure  glycerin, 


40      grains. 
15 

0'5  gram. 

0-2       „ 
40      grams. 


Filter,  distribute  in  flasks  of  250  c.c.  capacity  and  sterilize  at  115°  C. 

To  prepare  the  toxin  sow  the  medium  with  a  virulent  anthrax  bacillus 
and  incubate  for  48  hours  at  37°  C.  and  afterwards  at  20°  C.  for  a  fortnight. 

Now  filter  the  culture  through  porcelain  and  saturate  the  filtrate  with 
ammonium  sulphate  at  the  temperature  of  the  laboratory.  After  standing 
for  about  15  hours  filter  through  paper  and  wash  the  filter  with  a  saturated 
solution  of  ammonium  sulphate.  Wash  the  precipitate  which  remains  on 
the  filter  paper  with  as  small  a  quantity  of  glycerin  as  possible  :  leave  for 
2  days,  decant  the  glycerin,  replace  it  by  fresh  glycerin  and  decant  again. 
Mix  the  glycerin  solutions  together  and  add  the  mixture  to  four  times  its 
weight  of  strong  alcohol :  pour  the  precipitate  on  a  filter  and  wash  first  with 
absolute  alcohol,  then  with  ether  and  finally  dry  in  vacuo.  The  product  is 
an  amorphous,  easily  powdered  substance  dark  brown  in  colour  and  contains 
small  quantities  of  ammonium  sulphate.  It  is  soluble  in  distilled  water  and 
in  1  per  cent,  carbolic  acid.  It  has  none  of  the  properties  of  albuminoid 
substances,  peptones,  parapep tones  nor  alkaloids. 

This  substance  is  rather  toxic  for  rabbits  and  the  inoculation  of  small 
doses  may  cause  the  death  of  the  animal,  but  the  dose  varies  for  each  individual 
within  somewhat  wide  limits.  Thus  some  rabbits  succumb  to  the  inoculation 
of  25  mg.  (in  aqueous  solution)  while  for  others  the  fatal  dose  is  as  much  as 
120  or  even  200  mg. 

A  few  hours  after  inoculation  there  is  a  well-marked  rise  of  temperature  :  for  a 
few  days  the  temperature  oscillates  widely,  and  then  if  the  animal  is  going  to  die  it 
falls  steadily  (perhaps  as  low  as  8°  C.  below  normal),  if  on  the  other  hand  the  animal 
is  going  to  recover  the  temperature  oscillates  less  and  less.  The  animal  is  ill, 
cachectic,  and  may  lose  one-third  of  its  weight ;  as  a  rule  diarrhoea  is  a  symptom. 

2L 


530  THE  ANTHRAX  BACILLUS 

Before  death  symptoms  of  paraplegia  appear,  respiration  is  laboured  and  the  animal 
lies  on  its  side  and  may  have  convulsive  attacks. 

Death  may  take  place  at  any  time  between  the  2nd  and  15th  or  20th  day  after 
inoculation,  the  duration  of  life  depending  upon  the  amount  of  toxin  inoculated. 

Guinea-pigs  and  mice  are  susceptible  to  the  toxin. 

Contrary  to  Hankin's  experience  animals  immune  to  anthrax  are  almost 
unaffected  by  the  toxin  :  this  is  also  true  with  rabbits  immunized  with 
attenuated  cultures. 

The  toxin  is  weakened  but  not  completely  destroyed  by  heating  it  to 
110°  C.  The  addition  of  alkaline  hypochlorites,  chloride  of  gold,  or  Gram's 
solution  destroys  its  toxic  properties,  as  does  also  prolonged  exposure  to 
sunlight  in  presence  of  air. 

Cultures  of  anthrax  in  other  liquid  media  (ox  serum  or  broth  made  with  beef, 
veal  or  horse)  contain  but  little  toxin.  Marmier  was  able  to  extract  an  active 
toxin  from  recent  cultures  on  agar:  the  growth  from  48 -hour  old  cultures  on  agar 
was  scraped  and  macerated  in  alcohol  containing  a  few  drops  of  ether  at  20°  C.  ; 
after  autolyzing  for  24  hours  the  emulsion  was  filtered  and  the  filtrate  precipitated 
with  absolute  alcohol :  the  precipitate  on  the  filter  was  washed  with  absolute  alcohol 
then  with  ether  and  finally  dried  in  vacuo  over  sulphuric  acid.  The  powdery  product 
was  as  toxic  as  the  toxin  prepared  from  cultures  in  glycerin-peptone-water.  This 
indicates  that  primarily  the  toxins  are  intra- cellular. 

Vaccination  with  Toxin. 

I.  Toussaint   conferred   immunity   on   sheep   by   inoculating   them   with 
denbrinated  anthrax  blood  heated  to  55°  C.  for  10  minutes. 

Better  results  are  obtained  if  the  anthrax  blood  be  heated  to  60°  C.  on 
three  or  four  different  occasions  before  injecting  the  sheep.  A  slight  degree 
of  immunity  is  obtained  in  this  way  which  disappears  after  a  lapse  of  time 
varying  from  a  month  to  3  years  (Roux  and  Chamberland). 

II.  Hankin  stated  that  with  a  toxin  which  he  had  prepared  by  growing 
the  bacillus  in  broth  made  with  Liebig's  extract  and  containing  fibrin,  animals 
could  be  easily  immunized  against  anthrax.     After  certain  objections  had 
been  raised  by  Peterman,  Hankin  reinvestigated  the  subject  and  obtained 
less  satisfactory  results.     If  the  inoculation  of  toxin  in  amounts  equal  to 
i~Wooo  °f  k°dy  weight  has  any  immunizing  action  at  all  on  mice  it  must 
be  of  a  transitory  nature  and  in  any  case  only  a  small  proportion  of  animals 
treated  in  this  way  resist  the  test  inoculation. 

HI.  Marmier  succeeded  in  immunizing  laboratory  animals  with  a  toxin 
prepared  by  growing  the  bacillus  in  glycerin-peptone-water.  Rabbits  after 
being  repeatedly  inoculated  with  small  doses  acquired  a  certain  degree  of 
immunity  which  however  did  not  last  longer  than  5  or  6  weeks  after  the 
last  inoculation.  It  is  nevertheless  possible  to  immunize  rabbits  against 
doses  of  toxin  which  if  given  to  untreated  animals  would  cause  their  death. 

The  method  is  as  follows :  The  rabbit  is  first  of  all  inoculated  with  a  very  small 
dose  of  this  toxin,  for  example  3  mg.,  and  when  it  has  recovered,  that  is  to  say  in 
about  6  days,  a  larger  dose  (6  mg.)  is  administered  and  when  the  reaction  has  sub- 
sided a  third  dose  of  15  mg.  is  given.  About  12  days  after  the  third  inoculation  the 
animal  can,  in  the  majority  of  cases,  survive  the  inoculation  of  a  virulent  culture 
of  anthrax.  By  gradually  increasing  the  amount  inoculated  to  20  and  30  mg. 
immunity  can  almost  certainly  be  ensured.  Care  must  be  taken  to  see  that  the 
animal  has  completely  recovered  from  the  effects  of  the  immunizing  inoculations 
before  giving  the  test  inoculation. 

4.  Serum  therapy. 

I.  Behring  has  shown  that  the  serum  of  white  rats  exhibits  bactericidal 
properties  against  the  anthrax  bacillus.  When  a  small  quantity  of  a  culture 


SERUM  THERAPY  531 

of  the  anthrax  bacillus  is  mixed  with  some  rat  serum  and  inoculated  into 
mice,  the  mice  suffer  no  harm  (p.  518). 

Roux  and  Metchnikoff  have  proved  that  the  bactericidal  action  of  the  rat  serum 
is  only  exercised  when  the  serum  is  mixed  with  the  bacilli ;  if  the  culture  and  serum 
be  separately  inoculated,  the  mice  die  of  anthrax.  These  observers  also  showed 
that  the  high  degree  of  immunity  to  anthrax  usually  possessed  by  the  white  rat 
is  not  due  to  the  bactericidal  action  of  its  serum  for  they  found  that  a  large  number  of 
white  rats  whose  serum  was  known  to  be  bactericidal  were  susceptible  to  the  disease. 

II.  Marchoux  has  shown  that  the  serum  of  rabbits  and  sheep  immunized 
against  anthrax  by  means  of  attenuated  cultures,  while  possessing  both  pro- 
phylactic and  therapeutic  properties,  has  no  bactericidal  or  antitoxic  action. 

The  rabbits  should  be  immunized  by  the  method  described  at  p.  528.  Sheep 
after  being  vaccinated  by  Pasteur's  method  are  inoculated  with  larger  and  larger 
doses  of  virulent  cultures  at  intervals  of  a  week  until  an  amount  equal  to  200-300  c.c. 
is  given  in  one  inoculation.  The  sheep  must  be  able  to  withstand  these  enormous 
doses  before  their  serums  become  prophylactic  or  curative.  The  animal  is  bled 
15  or  20  days  after  the  last  protective  inoculation  as  experience  has  shown  that 
the  serum  is  most  potent  at  this  period.  The  serum  retains  its  properties  for  a 
long  time. 

(a)  Marchoux   obtained  a  sheep  serum  which  had  a  titre  of  ^QQQ  (1  c.c. 
inoculated  24  hours  before  0'25  c.c.  of  a  virulent  culture  protected  a  rabbit 
weighing  2  kg.).     The  inoculations  were  made  beneath  the  skin  of  the  flank, 
the  serum  on  one  side  the  culture  on  the  other. 

Inoculation  of  culture  beneath  the  skin  of  the  ear  has  more  severe  results, 
and  to  protect  the  animal  twice  the  quantity  of  serum  used  in  the  preceding 
case  must  be  given.  Inoculation  of  culture  into  the  peritoneum  requires  a 
still  larger  protective  dose  of  serum,  at  least  15  c.c.  of  a  2-00Q  serum  being 
required  to  protect  a  rabbit  weighing  2  kg.  When  the  culture  is  administered 
intra-venously  20  c.c.  of  a  —&  semm  OT^J  prolongs  the  life  of  the  rabbit 
for  3  days  beyond  that  of  the  control. 

The  serum  is  equally  effective  whether  inoculated  intra-peritoneally  or 
sub-cutaneously.  Intra- venous  injection  however  appears  to  be  less  effective 
since  10  c.c.  of  a  2"Q00  serum  does  not  protect  a  rabbit  weighing  2  kg.  against 
the  sub-cutaneous'  inoculation  of  0'25  c.c.  of  a  virulent  culture. 

Note. — A  3,006  serum  has  no  prophylactic  properties  for  guinea-pigs.  Even 
by  using  very  large  doses  of  serum  Marchoux  was  able  only  to  prolong  life  and 
that  but  for  a  variable  length  of  time. 

(b)  The  serum  of  a  vaccinated  rabbit  inoculated  at  the  same  time  as  a 
virulent  culture  ensured  the  recovery  of  the  animal  (rabbit)  in  7  out  of  24 
experiments  :   in  the  remaining  17  cases  the  animal  always  lived  longer  than 
the  controls.     The  amount  of  serum  inoculated  varied  from  7-17  c.c.     The 
rabbits  which  survived  showed  no  symptom  of  illness  while  all  those  in  which 
an  oedema  developed,  died. 

(c)  A  rabbit  which  was  given  6  c.c.  of  the  serum  of  a  vaccinated  rabbit 
4  hours  after  the  inoculation  of  bacilli  survived.     And  another  in  which 
the  serum  was  administered  7  hours  after  the  inoculation  of  bacilli  died 
108  hours  after  the  control. 

With  a  -g^-fj  sheep  serum  Marchoux  was  able  to  cure  a  rabbit  inoculated 
7  hours  previously  (dose,  7  c.c.  of  serum).  With  a  2-J00  serum  the  inoculation 
of  10  c.c.  given  24  hours  after  the  culture  was  followed  by  recovery. 

If  at  the  moment  of  inoculation  of  the  serum  there  is  a  well-marked  oedema 
recovery  does  not  take  place  even  though  relatively  enormous  doses  of  serum 
be  used  (e.g.  15-20  c.c.  of  a  2^  serum). 


532  THE   ANTHRAX   BACILLUS 

Note. — When  the  serum  is  inoculated  before  or  immediately  after  a  virulent 
culture,  the  rabbit  shows  no  signs  of  illness  but  it  has  acquired  no  immunity  against 
the  bacillus  :  for,  if  inoculated  later,  it  succumbs  to  anthrax  after  the  same  lapse 
of  time  as  control  animals.  On  the  other  hand,  when  there  has  been  some  delay 
so  that  the  serum  is  not  given  for  7-24  hours  after  the  infection  the  animal  has 
become  sufficiently  ill  to  enable  it  to  acquire,  during  recovery  and  with  the  aid  of  the 
serum,  a  marked  resistance  to  anthrax. 

III.  Sclavo  undertook  a  series  of  experiments  which  enabled  him  to  pro- 
duce serums  endowed  like  that  of  Marchoux  with  prophylactic  and  therapeutic 
properties  but  devoid  of  all  bactericidal  or  antitoxic  properties. 

(a)  Sheep  after  being  first  treated  with  the  two  vaccines  of  Pasteur  were 
then  repeatedly  inoculated  with  virulent  cultures  in  gradually  increasing 
quantities.     A  serum  was  thus  obtained  which  in  doses  of  2  c.c.  protected 
rabbits  against  doses  of  anthrax  fatal  to  animals  not  so  treated. 

(b)  Asses  are  still  more  useful  for  the  preparation  of  antianthrax  serum. 
Ottolenghi   prepared   an   ass    serum  by  Sclavo's   method   which  protected 
guinea-pigs  against  a  very  virulent  virus  provided  that  it  was  administered 
intra-peritoneally  24  hours  before  the  test  inoculation. 

(c)  It  is  easy  to  secure  a  sero-vaccination  in  guinea-pigs  by  inoculating 
6  c.c.  of  immunized  ass  serum  sub-cutaneously  and  1  c.c.  of  Pasteur's  first 
vaccine  intra-peritoneally. 

IV.  Sobernheim,  repeating  and  extending  Sclavo's  work  immunized  sheep 
by  inoculating  them  with  10  c.c.  of  serum  intra-venously  and  with  a  culture 
of  anthrax  sub-cutaneously  :    if  sheep  immunized  in  this  way  be  repeatedly 
inoculated  with  virulent  cultures  a  very  active  serum  is  obtained. 

Sobernheim  succeeded  in  conferring  a  substantial  immunity  on  horses  and 
sheep  in  10-12  days.  The  animals  were  inoculated  with  5  c.c.  of  serum  and 
0'25-0'5  c.c.  of  a  culture  of  anthrax  sub-cutaneously  in  different  parts  of  the 
body.  According  to  Sobernheim  the  successful  use  of  the  serum  depends 
above  all  in  using  it  in  combination  with  the  virus. 

V.  Sanfelice  prepared  a  dog  serum  having  very  marked  prophylactic  and 
therapeutic  properties.     The  dogs  were  first  of  all  inoculated  sub-cutaneously 
with  cultures  attenuated  by  growing  them  for  5-7  days  at  a  temperature 
of  37°  C.   and  subsequently  with  more  and  more  virulent  cultures.     The 

>rocess  of  immunization  lasted  about  a  month.  The  serum  was  then  found 
to  be  very  powerfully  prophylactic  for  rabbits  but  not  for  guinea-pigs.  It 
possessed  neither  bactericidal  or  antitoxic  properties  but  nevertheless  proved 
of  value  as  a  therapeutic  agent :  in  doses  of  7  c.c.  per  1  kg.  of  body  weight 
it  arrested  the  infection  in  rabbits  if  given  within  40  hours  of  the  inoculation 
of  bacilli.  If  the  delay  were  greater  than  this  death  ensued  whatever  the 
dose  employed. 

In  a  man  infected  with  anthrax  Sanfelice  observed  that  the  symptoms 
abated  on  the  third  day  after  the  inoculation  of  56  c.c.  of  serum. 

[Human  serum  therapy. — Immune  ass  serum  prepared  by  Sclavo's  method 
has  been  used  in  the  treatment  of  anthrax  in  man  since  1897  with  distinctly 
encouraging  results. 

[The  inoculations  are  administered  sub-cutaneously.  The  first  inoculation 
consists  of  a  dose  of  antianthrax  serum  followed  the  next  day  by  a  dose  of  a  broth 
culture  of  the  second  vaccine  ;  ten  days  later  an  inoculation  consisting  of  a  mixture 
of  antianthrax  serum  and  virulent  anthrax  bacilli  is  given  and  then  at  more  or  less 
regular  intervals  of  10  days  gradually  increasing  doses  of  virulent  anthrax  bacilli 
without  serum  until,  when  tested  on  rabbits,  it  protects  these  animals  from  a  dose 
of  living  virulent  bacilli  sufficient  to  kill  control  rabbits.  The  ass  is  bled  to  the 
extent  of  about  150  c.c.  after  a  suitable  interval  from  the  last  inoculation  and  the 
immunity  is  maintained  by  the  inoculation  of  young  gelatin-broth  cultures  sub- 
cutaneously  at  intervals. 


SERUM   THERAPY  533 

[For  the  treatment  of  anthrax  in  man  the  initial  dose  of  the  serum  should 
be  large — 40  c.c. — and  may  be  repeated  if  necessary.  The  serum  is  generally 
inoculated  sub-cutaneously  but  in  severe  cases  intra-venously.  As  with  all 
other  therapeutic  serums  the  sooner  it  is  administered  after  the  infection  has 
taken  place  the  better  will  be  the  result  of  the  treatment. 

[The  immediate  results  following  inoculation  of  the  serum  are  sometimes 
rather  startling  ;  the  temperature  rises  often  to  over  105°  F.  and  the  patient 
becomes  very  ill,  at  the  same  time  the  local  lesion  appears  to  get  much  worse 
and  the  oedema  increases.  Then  follows  a  period  of  recovery  during  which 
the  temperature  falls  to  normal,  the  size  of  the  inflamed  area  diminishes  and 
the  bacilli  disappear  from  the  lesion. 

[The  rapid  disappearance  of  bacilli  from  the  lesions  is  one  of  the  most  striking 
results  of  serum  treatment.  In  one  of  the  first  cases  treated  in  England  Andrewes 
found  that  no  bacilli  could  be  cultivated  from  the  fluid  of  the  vesicles  19  hours 
after  the  administration  of  40  c.c.  of  serum  though  previously  abundant  colonies 
developed  on  the  medium  sown  with  the  fluid. 

["  The  claims  which  have  been  made  as  to  the  effect  of  serum  treatment 
can  be  summarized  by  saying  that :  (1)  even  in  very  large  doses  it  is  innocu- 
ous ;  (2)  it  can  be  well  borne  even  when  introduced  into  the  veins  ;  (3)  no 
case  taken  in  an  early  stage  or  of  moderate  severity  is  fatal  if  treated  with 
serum  ;  (4)  with  the  serum  some  cases  are  saved  when  the  condition  is  most 
critical  and  prognosis  almost  hopeless  ;  (5)  when  injected  into  the  veins  the 
serum  quickly  arrests  the  extension  of  the  oadematous  process  so  as  to  reduce 
notably  the  danger  from  suffocation  which  exists  in  many  of  the  cases  where 
the  pustule  is  situated  on  the  face  or  neck  ;  (6)  if  used  soon  enough  it  reduces 
to  a  minimum  the  destruction  of  the  tissues  at  the  site  of  the  pustule  ;  (7)  in 
some  situations  of  the  pustule,  as  the  eyelid,  serum  treatment  must  be  used 
in  preference  to  any  other  as  it  alone  can  hold  out  hope  of  success  without 
permanent  injury  ;  and  (8)  in  internal  anthrax  it  is  the  only  treatment  which 
holds  out  any  hope  of  benefit  "  (Legge). 

[In  1903  Sclavo  tabulated  all  cases  known  to  have  been  treated  with  his 
serum  up  till  that  time  in  Italy.  In  all  164  cases  were  treated  with  10  deaths 
—a  rate  of  6'09  per  cent,  as  against  24'1  per  cent,  for  the  whole  of  Italy.] 

5.  Agglutination. 

Cultures  of  the  anthrax  bacillus  do  not  lend  themselves  to  the  demonstra- 
tion of  the  phenomena  of  agglutination  because  the  bacilli  are  linked 
together  in  chains.  To  study  the  phenomenon  it  is  necessary  to  work  with 
attenuated  cultures  (and  especially  Pasteur's  No.  1  vaccin)  with  which  fairly 
homogeneous  emulsions  can  be  obtained.  These  emulsions  are  agglutinated 
by  the  serum  of  various  untreated  animals  (rat,  rabbit,  guinea-pig,  ox,  horse, 
etc.)  in  dilutions  of  1  in  10  to  1  in  50.  Normal  human  serum  agglutinates 
them  powerfully — even  in  dilutions  of  1  in  500  in  some  cases.  "  Great  care 
should  be  exercised  in  the  serum  diagnosis  of  anthrax  "  (Lambotte  and 
Marechal).  Sobernheim  arrived  at  a  similar  conclusion  namely  that  there  is 
no  relation  between  the  degree  of  agglutination  reaction  and  the  degree  of 
immunization  :  sometimes  a  normal  serum  will  agglutinate  as  powerfully  as 
a  serum  obtained  from  immunized  animals. 

SECTION  IV.— DETECTION,   ISOLATION  AND   IDENTIFICATION   OF 
THE   ANTHRAX   BACILLUS. 

The  anthrax  bacillus  should  be  looked  for— 

I.  In  fche  serous  fluid  of  the  malignant  pustule  in  man  and  in  the  gelatinous 
cedema  in  the  lower  animals. 


534  THE   ANTHRAX   BACILLUS 

2.  In  the  blood  and  urine,  and  in  smears  and  sections  of  organs,  in  both 
man  and  the  lower  animals. 

The  clinical  diagnosis  of  malignant  pustule  in  man  should  always  be  con- 
firmed by  bacteriological  investigation  ;  and  in  any  suspected  or  ascertained 
case  of  the  disease  the  blood  and  urine  should  be  immediately  examined 
microscopically  since  in  both  man  and  the  lower  animals  the  entrance  of  the 
bacillus  into  the  blood  stream  denotes  generalization  of  the  infection  and  a 
fatal  termination,  death  occurring  soon  after  its  appearance. 

The  blood  in  the  Jiving  human  subject  should  be  obtained  by  pricking  the  finger 
or  the  lobe  of  the  ear.  In  the  case  of  animals  the  blood  is  best  collected  from 
the  ear. 

Serum  from  the  malignant  pustule  may  be  obtained  by  scratching  the  surface 
of  the  pustule  with  a  lancet  after  sterilizing  the  skin  with  antiseptics. 

Other  material  can  be  collected  in  the  ordinary  way. 

To  make  a  complete  bacteriological  examination  it  is  necessary  to  confirm 
the  microscopical  examination  by  sowing  cultures  and  inoculating  animals. 

(a)  Cultures. — Sow  the  material  (blood,  scraping  from  organs,  exudates 
etc.)  on  ordinary  media  and  incubate  aerobically. 

(6)  Inoculations. — Inoculate  a  guinea-pig  (for  preference)  or  a  mouse  sub- 
cutaneously  with  a  few  drops  of  blood,  or  with  a  scraping  from  one  of  the 
internal  organs  rubbed  up  in  sterile  water,  or  preferably  with  a  24-hour 
growth  obtained  by  sowing  the  medium  with  blood  or  other  material. 

(c)  Microscopical  examination. — For  microscopical  examination  prepare  : 

1.  Blood  films. 

2.  Smears  of  the  internal  organs  and  especially  of  the  spleen. 

3.  Smears  from  the  gelatinous  cedema  or  exudate  from  the  malignant  pustule. 

4.  Mesentery. — This  is  best  examined  in  the   mouse.     Remove  a  small 
piece  of  the  mesentery,  spread  it  out  on  a  slide  with  needles  leaving  the 
edges  to  dry  a  little  so  as  to  assure  its  adhering  to  the  glass,  then  pull  on  it 
in  such  a  manner  as  to  stretch  the  membrane,  treat  with  alcohol-ether  then 
stain. 

5.  Sections  of  tissues. — Cut  off  small  pieces  of  the  liver,  spleen,  lungs,  and 
kidneys,  fix  in  absolute  alcohol  and  embed  in  paraffin.     Stain  as  indicated 
above  (p.  522). 

The  examination  of  carcases  dead  of  the  spontaneous  disease. 

When  examining  material  from  a  dead  body  it  should  be  remembered  that 
anthrax  carcases  very  soon  after  death  become  invaded  by  the  bacillus  of 
malignant  oedema  (Chap.  XXXVIII.),  a  micro-organism  which  on  superficial 
examination  is  liable  to  be  confounded  with  the  anthrax  bacillus  ( Jaillard  and 
Leplat). 

[In  England  the  carcase  of  an  animal  suspected  to  have  died  from  anthrax 
may  not  be  opened.  For  bacteriological  examination  the  Board  of  Agriculture 
require  that  an  ear  shall  be  cut  off. 

[The  ear  should  be  pinned  out  on  a  board  and  the  outer  surface  washed  with 
2  per  cent,  lysol.  An  incision  should  then  be  made  with  a  sterile  scalpel  from  the 
base  to  the  tip  and  the  skin  reflected  from  the  subjacent  cartilage.  With  another 
sterile  knife  one  of  the  small  veins  thus  exposed  should  be  cut  across  an  inch  or 
so  from  the  base — to  avoid  organisms  which  may  have  contaminated  the  cut  sur- 
face— and  the  blood  squeezed  out.  Films  are  then  prepared  and  stained  (p.  522) 
and  cultures  sown. 

[It  is  of  importance  to  recognize  that  the  mere  microscopical  examination 
of  blood  films  cannot  be  relied  upon  for  the  recognition  of  anthrax  in  animals 
suspected  to  have  died  of  the  disease.  It  is  true  that  in  the  great  majority 
of  cattle  which  have  died  of  anthrax,  bacilli  can  be  seen  in  large  numbers  in 


THE  ISOLATION  OF  THE   BACILLUS  535 

blood  films  prepared  as  described  above  but  there  is  a  minority  of  cases  in 
which  anthrax  exists  but  in  which  the  bacilli  cannot  be  found  on  microscopical 
examination  because  the  organisms  are  present  only  in  very  small  numbers  in 
the  blood.  In  horses,  and  especially  in  pigs,  a  negative  result  on  micro- 
scopical examination  is  of  no  value.  Agar  cultures  must  be  sown  therefore 
in  every  suspected  case  of  anthrax  in  animals.  If  bacilli  resembling  anthrax 
bacilli  were  found  in  the  blood  films,  examination  of  the  cultures  will  confirm 
the  diagnosis ; — the  bacillus  of  malignant  oedema  does  not  grow  on  agar 
under  aerobic  conditions.  In  those  cases  in  which  no  bacilli  were  found  in  the 
blood  films  and  cultures  yield  an  organism  resembling  the  anthrax  bacillus 
the  diagnosis  must  be  confirmed  by  the  inoculation  of  a  guinea-pig.] 

Cin£a  and  Stoiesco  have  shown  that  it  is  of  great  advantage  to  examine 
the  skin  in  cases  where  the  carcase  has  'begun  to  decompose.  The  anthrax 
bacillus  can  always  be  found  in  the  skin  even  though  it  may  have  disappeared 
from  the  blood  and  internal  organs. 

Cut  off  small  pieces  of  the  skin  and  leave  them  to  dry,  then  scrape  them  with  a 
scalpel  and  rub  up  the  scrapings  with  sterile  saline  solution  (10  volumes  of  saline  to 
1  volume  of  tissue).  To  a  series  of  broth  tubes  add  1—5  drops  of  the  emulsion,  heat 
the  tubes  to  65°  C.  for  thirty  minutes,  then  sow  on  agar,  and  examine  and  identify 
the  colonies  which  develop. 

Examination  of  the  intestinal  contents  may  also,  according  to  Cin9a  and 
Fenea,  afford  valuable  evidence  in  diagnosis. 

Isolation  of  the  bacillus  from  soil. 

Pasteur  utilized  the  resistance  of  the  spores  to  heat  for  the  purpose  of  isolating 
the  organism  from  the  soil  of  infected  fields.  The  technique  is  as  follows  : 

Crush  a  little  of  the  soil  in  a  mortar  and  make  a  suspension  in  sterile  water.  A 
granular  precipitate  is  immediately  deposited.  Decant  the  supernatant  liquid 
containing  only  fine  light  particles  with  care  and  let  it  settle  in  a  sterile  test-tube 
on  a  foot.  The  turbid  liquid  becomes  clear  and  a  deposit  is  formed  in  the  bottom 
of  the  tube.  Decant  the  supernatant  liquid  again.  Aspirate  the  deposit  into 
pipettes  and  after  sealing  heat  them  in  a  water-bath  at  85°  C.  for  15-20  minutes  and 
use  the  contents  for  sowing  gelatin  plates  in  Petri  dishes.  The  plates  must  be 
carefully  watched,  and  every  suspicious  colony  should  be  examined  microscopically, 
sown  on  various  media,  and  inoculated  into  guinea-pigs  and  mice. 

The  pyogenic  micro-organisms  are  destroyed  by  heating  the  material  to  85°  C. 
and  the  method  of  cultivation  eliminates  all  anaerobic  species  such  as  the  bacillus 
of  malignant  oedema  which  might  lead  to  error  in  the  investigation.  In  the  method 
originally  used  by  Pasteur  the  deposit  obtainr  by  powdering  the  soil  was  inoculated 
immediately  after  heating  and  before  colonies  were  isolated,  but  this  technique  is 
not  so  exact. 

By  utilizing  a  method  very  similar  to  that  just  described,  Diatroptoff  succeeded 
in  isolating  the  organism  from  the  mud  at  the  bottom  of  a  well. 


CHAPTEE  XXXVI. 
BACILLUS   TETANI. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  536. 

1.  Inoculation  of  soil  or  pus,  p.  537.     2.  Inoculation  of  pure  cultures,  p.  537. 

3.  Inoculation  of  spores,  p.  538. 
Section  II. — Morphology,  p.  539. 

1.  Microscopical  appearance  and  staining  reactions,  p.  539.     2.  Cultural  charac- 
teristics, p.  540. 
Section  III. — Biological  properties,  p.  541. 

1.  Vitality  and  virulence,  p.   541.     2.  Toxin,  p.   541.     3.  Vaccination,  p.  544. 

4.  Serum  therapy,  p.  545.     5.  Agglutination,  p.  548. 

Section  IV. — Detection,  isolation  and  identification  of  the  tetanus  bacillus,  p.  548. 

Bacillus  botulinus. 

TETANUS  is  a  disease  which  affects  not  only  man  but  all  species  of  domestic 
animals  and  is  due  to  the  bacillus  of  tetanus  discovered  by  Nicolaier. 

Tetanus  in  horses,  asses,  cows,  etc.  generally  follows  wounds  of  the  foot.  Tetanus 
has  been  known  to  affect  a  number  of  horses  in  an  almost  epizootic  manner  after 
castration,  the  organism  in  these  cases  being  conveyed  by  the  instruments  employed 
in  the  operation. 

Tetanus  in  man  is  known  to  follow  accidental  wounds  or  surgical  operations :  the 
organism  having  been  introduced  at  the  time  of  the  injury. 

Much  has  also  been  said  about  idiopathic  tetanus  which  develops  in  the  apparent 
absence  of  all  solution  of  continuity  of  the  integuments.  These  so-called  idiopathic 
cases  are  due  either  to  an  infection  '.  rough  the  alimentary  canal  (Siisse)  or  more 
likely  to  the  introduction  of  the  organism  some  time  previously  through  a  wound 
which  has  since  healed  and  been  forgotten.  Vaillard  and  Rouget  have  shown  that 
spores  introduced  into  the  tissues  can  remain  dormant  for  a  very  long  time,  ger- 
minating when  the  conditions  become  favourable,  and  thus  setting  up  a  disease 
which  would  appear  to  be  spontaneous. 

The  spores  of  the  tetanus  bacillus  are  very  widely  distributed  outside  the 
body.  If  a  guinea-pig  be  inoculated  with  garden  soil,  or  with  dust  or  mud 
from  the  street  it  almost  always  dies  either  from  tetanus  or  malignant  oedema 
(Nicolaier).  The  tetanus  bacillus  is  also  found  in  the  contents  of  the  large 
gut  and  in  the  excreta  of  many  animals. 

SECTION  I.— EXPERIMENTAL   INOCULATION. 

Mice,  rats  and  guinea-pigs  are  very  susceptible  to  tetanus,  rabbits  less  so, 
while  dogs  are  very  difficult  to  infect,  and  pigeons  and  fowls  are  naturally 
immune  to  the  disease. 

Susceptible  animals  may  be  infected  in  many  ways. 


EXPERIMENTAL  INOCULATION  537 

• 

1.  By  inoculation  of  specifically  infected  pus  from  a  wound  in  man  or  the 
lower  animals. 

2.  By  inoculation  of  soil. 

3.  By  inoculation  of  a  pure  culture  of  the  bacillus. 

4.  By  inoculation  of  the  spores  from  a  culture  of  the  organism. 

5.  By  inoculation  of  tetanus  toxin  (vide  infra). 

Whatever  the  mode  of  infection  the  tetanus  bacillus  never  invades  the 
tissues  of  the  organism  but  remains  strictly  localized  at  the  site  of  inoculation. 

When  animals  have  been  inoculated  intra-venously  or  into  the  peritoneum  with 
a  large  dose  of  culture  the  bacillus  may  be  found  in  cultures  sown  with  the  blood 
and  internal  organs.  Sanchez  Toledo  and  Veillon  occasionally  obtained  a  growth 
of  the  bacillus  from  the  blood  of  an  animal  dead  of  tetanus  by  allowing  a  certain 
time  to  elapse  after  death  before  sowing  the  cultures.  But  these  results  are  quite 
exceptional. 

A  few  hours  after  inoculation  the  bacilli  have  diminished  in  number  and 
it  is  not  long  before  their  presence  at  the  site  of  injection  can  only  be  demon- 
strated by  culture  methods.  If  a  minimal  lethal  dose  be  inoculated,  the 
material  collected  post  mortem  from  the  infected  part  is  not  infectious  on 
reinoculation.  On  the  other  hand,  pus  from  an  infected  wound  will  convey 
the  disease  to  susceptible  animals  but  it  is  impossible  to  effect  more  than 
four  passages  from  animal  to  animal.  The  second  passage  animal  does  not 
die  so  quickly  as  the  first  and  so  on  ;  at  the  fourth  passage  the  virulence  of 
the  bacillus  is  greatly  diminished. 

1.  Inoculation  of  soil  or  infected  pus. 

The  inoculation  is  best  effected  either  beneath  the  skin  or  into  the  muscles 
of  the  thigh  of  a  guinea-pig  or  mouse. 

Symptoms  and  lesions. — A  swelling  forms  at  the  site  of  inoculation  and 
the  part  is  puffy  and  painful ;  3  or  4  days  after  the  inoculation  symptoms 
of  tetanus  appear  beginning  in  the  neighbourhood  of  the  infected  area,  and 
becoming  generalized :  the  slightest  stimulus,  such  as  a  noise,  a  draught  of 
air,  a  touch  etc.,  will  produce  spasmodic  movements.  The  clinical  condition 
is  exactly  the  same  as  in  human  tetanus.  Death  occurs  24—48  hours  after 
the  onset  of  the  symptoms. 

Post  mortem  :  at  the  site  of  inoculation  there  will  either  be  a  purulent  focus, 
or  a  lesion  resembling  a  dry  yellow  slough,  or  a  thick  membranous  exudate. 
The  neighbouring  tissues  are  the  site  of  an  oedematous  infiltration.  On 
microscopical  examination  numerous  other  micro-organisms — one  or  two 
species  predominating — will  be  found  associated  with  the  tetanus  bacillus. 
The  purulent,  membranous  and  necrotic  lesions  are  due  to  organisms  other 
than  the  tetanus  bacillus.  The  internal  organs  are  healthy  and  merely  show 
a  slight  congestion  due  to  the  embarrassed  respiration  which  precedes  death. 

2.  Inoculation  of  pure  cultures. 

Infection  may  be  set  up  by  inoculating  pure  cultures  sub-cutaneously, 
intra-muscularly,  intra-peritoneally,  intra-venously,  sub-durally  or  on  to  the 
ocular  conjunctiva.  Ingestion  is  the  only  mode  of  introduction  which  fails 
to  produce  the  disease.  Sub-cutaneous  or  intra-muscular  inoculation  is  the 
most  certain  and  rapid  method  of  infecting  an  animal. 

Very  small  doses  of  broth  cultures  suffice  to  set  up  the  disease  in  susceptible 
animals.  0'02  c.c.  will  produce  a  typical  tetanus  in  mice  and  guinea-pigs  : 
the  symptoms  begin  12-20  hours  after  inoculation  and  terminate  fatally  in 
36-40  hours. 

In  the  case  of  rabbits,  a  dose  of  0'5-1'5  c.c.  is  necessary  and  even  then  the 


538  THE   TETANUS   BACILLUS 

• 

earliest  symptoms  do  not  appear  until  between  the  second  and  eighth  day 
and  death  does  not  occur  until  3-10  days  after  the  onset  of  the  symptoms. 

Symptoms  and  lesions.— Whatever  the  dose  inoculated  there  is,  before 
symptoms  appear,  a  period  of  incubation  the  length  of  which  varies  with 
the  virulence  of  the  culture,  the  dose  inoculated  and  the  resistance  of  the 
animal.  As  a  general  rule,  the  severity  of  the  disease  varies  inversely  as 
the  incubation  period — the  shorter  the  incubation  period  the  more  severe 
and  more  rapidly  fatal  the  disease.  If  the  incubation  period  exceed  5  days 
in  guinea-pigs  and  8  days  in  rabbits,  the  disease  assumes  the  chronic  type, 
lasts  some  10-30  days  and  may  end  in  the  recovery  of  the  animal. 

As  in  the  previous  case  the  symptoms  always  begin  in  the  neighbourhood 
of  the  inoculation  and  then,  if  the  dose  be  sufficient,  become  generalized. 
If  the  dose  be  very  small  the  symptoms  may  be  limited  to  the  limb  or  group 
of  muscles  affected  by  the  inoculation. 

Post  mortem. — Beyond  the  occasional  occurrence  of  a  little  hypersemia  or 
a  slight  oedema,  both  very  circumscribed,  there  is  no  lesion  at  the  site  of 
inoculation.  Neither  can  any  lesions  be  found  in  the  internal  organs. 

Cultures  of  the  tetanus  bacillus  do  not  multiply  when  inoculated  into  the 
living  tissues,  but,  on  the  other  Hand,  the  organism  quickly  disappears. 

Cultures  filtered  through  a  Chamberland  bougie  produce  the  same  symptoms 
as  unfiltered  cultures.  This  matter  will  be  dealt  with  later,  here  it  is 
sufficient  to  point  out  that  the  results  obtained  by  inoculating  cultures  are 
due  to  the  toxin  they  contain. 

3.    Inoculation  Of  Spores  alone  (Vaillard,  and  Vincent  and  Rouget). 

If  a  broth  culture  of  the  tetanus  bacillus  containing  spores  be  heated  at 
80°  C.  for  3  hours,  the  toxin  is  destroyed  and  the  broth  contains  only  spores 
which  remain  unaffected  by  the  heating  process. 

Doses  of  0*5  or  0'6  c.c.  of  these  heated  cultures  can  be  inoculated  into 
guinea-pigs  without  the  animal  showing  any  symptoms  of  tetanus.  Pure 
spores  do  not  germinate  in  the  living,  healthy  tissues  and  cannot  therefore 
manufacture  the  toxin  necessary  to  produce  the  symptoms  of  the  disease. 
Spores  inoculated  in  the  pure  state  are  rapidly  ingested  and  digested  by  the 
phagocytes. 

If  however  a  negatively  chemiotactic  substance,  such  for  instance  as  a 
little  drop  of  lactic  acid,  be  mixed  with  the  spores  before  inoculation  the 
leucocytes  are  unable  to  approach  the  spores  which  now,  left  to  themselves, 
quickly  germinate  with  the  result  that  symptoms  of  tetanus  appear. 

The  same  result  may  be  arrived  at  by  mechanically  protecting  the  spores  against 
the  attacks  of  the  leucocytes.  For  instance,  if  the  spores  be  mixed  with  a  little  sterile 
sand  and  wrapped  in  a  small  piece  of  previously  sterilized  filter  paper,  the  paper 
envelope  constitutes  a  defence  which  the  leucocytes  cannot  penetrate ;  the  spores 
therefore  are  free  to  germinate  and  manufacture  toxin  with  the  result  that  the 
animal  dies  of  tetanus. 

Again,  if  an  injury  be  produced  at  the  site  of  inoculation,  such  as  a  burn,  or  a 
traumatism  of  the  tissues,  etc.  phagocytosis  is  interfered  with,  the  leucocytes 
cannot  attack  the  spores,  and  the  latter  develop  with  the  result  that  the  animal  will 
suffer  from  tetanus. 

Numerous  cases  of  tetanus  have  been  recorded  following  the  hypodermic  injec- 
tion of  quinine.  Vincent  has  shown  that,  like  lactic  acid,  the  salts  of  quinine  favour 
the  germination  of  tetanus  spores.  In  a  "  carrier  "  of  latent  tetanus  spores  an 
injection  of  quinine  will  be  followed  by  a  multiplication  of  tetanus  bacilli  at  the 
site  of  inoculation  of  the  quinine. 

Equally  interesting  and  important  in  the  aetiology  of  tetanus  is  the  role  of 
ancillary  organisms.  When  an  animal  succumbs  after  the  inoculation  of 


MORPHOLOGY 


539 


soil  containing  the  tetanus  bacillus,  other  organisms  in  addition  to  the  latter 
are  found  in  the  pus.  Vaillard  and  Rouget  isolated  several  of  these  organisms 
and  obtained  cultures  which  when  mixed  with  pure  spores  assisted  in  the 
development  of  the  disease  just  as  would  the  addition  of  a  negatively  chemio- 
tactic  substance.  Soil  containing  spores  of  tetanus  only  gives  rise  to  the 
disease  provided  that  it  contains  such  ancillary  organisms. 

Experiment. — Take  a  little  soil  containing  tetanus  spores  and  divide  it  into  two 
portions,  one  of  which  acts  as  the  control.  Mix  the  other  portion  with  sterile  water 
and  aspirate  it  into  a  pipette  as  far  as  the  constriction.  Seal  the  pipette  and  heat 
the  contents  at  85°  C.  for  an  hour.  The  spores  of  tetanus  can  survive  this  temperature 
while  non-sporing  organisms  are  destroyed.  If,  now,  some  of  the  unheated  soil 
be  inoculated  into  guinea-pigs,  the  animals  will  die  of  tetanus,  while  guinea-pigs 
inoculated  with  the  same  amount  of  heated  soil  will  be  unharmed. 

On  the  other  hand  cultures  sown  aerobically  with  the  same  (unheated)  soil  and 
inoculated  into  guinea-pigs  give  rise  to  purulent  lesions  but  never  to  tetanus.  A 
third  series  of  guinea-pigs  may  be  inoculated  with 

a  little  of  the  heated  soil  to  which  a  small  amount  f 

of  an  aerobic  culture  has  been  added.     In  this  v 

case  all  the  animals  will  die  of  tetanus.  >J 


SECTION  II.—  MORPHOLOGY. 


1.  Microscopical  appearance. 

The  bacillus  of  tetanus  occurs  sometimes  A 

as  spores,  sometimes  in  the  non-spore-bearing  ^x 

condition.  • 

A.  In  young  cultures,  and  in  some  cases  in 

TYII«     flip   nrrrnni«m    a<a«nmPQ    f>iP    fnrrn    nf    IT-PT-U-         FlG.  259. — Bacillus  tetani.     Pus  from 

pus,  tne  organism  assumes  tne  torm  ol  very  a  guinea.pig  showing  a  double  infection 
slender,  elongated  rods  with  square-cut  ends  with  a  coccus.  Carboi-biue.  (Oc.  n, 
measuring  3-4/u  by  0'3-0'4/K,  and  showing  in  c 

the  absence  of  oxygen  slow  wavy  movements  which  cease  when  the  bacillus 
becomes  spore-bearing. 

The  non-spore-bearing  bacillus  is  flagellated.  The  flagella  are  numerous, 
wavy  and  long  and  are  attached  laterally  to  the  body  of  the  bacillus  (peri- 
trichous)  (fig.  261).  They  can  be  readily  stained  by  the  usual  methods 
(p.  148). 


r 


Fm.  260. — Bacillus  tetani.  Broth  culture 
showing  spores.  Carbol-fuchsin  and  methy- 
lene  blue.  (Oc.  2,  obj.  TUh,  Zeiss.) 


FIG.  261. — Bacillus  tetani.     Showing 
flagella.      x  1000. 


B.  Spore-bearing  bacilli  will  be  found  in  cultures  incubated  at  37°  C.  for 
36-48  hours  and  sometimes  in  pus.     Cultures  10  days  old  consist  almost 


540  THE   TETANUS   BACILLUS 

entirely  of  bacilli  with  spores.  In  cultures  incubated  at  20°  C.  or  25°  C., 
spore  formation  is  slower  and  only  commences  after  the  cultures  have  been 
incubated  for  about  10  days. 

Spore-bearing  bacilli  occur  as  rather  short,  slender  rods  with  a  small  sphere 
situated  exactly  at  one  end  :  the  spore  is  refractile  and  has  a  diameter  two 
to  four  times  the  width  of  the  bacillus.  This  is  known  as  the  pin  form  of 
the  bacillus  (fig.  260). 

In  old  cultures,  the  body  of  the  bacillus  breaks  off  and  spores  and  swollen, 
irregular,  dumb-bell  shaped  involution  forms  only  are  found. 

Staining  reactions. — The  bacillus  of  tetanus  is  easily  stained  with  the 
basic  aniline  dyes,  and  is  gram-positive.  If  spore-bearing  bacilli  be  stained 
in  the  ordinary  way  the  rods  and  the  outline  of  the  spores  alone  are  stained, 
leaving  the  centres  of  the  latter  unstained  and  giving  the  organism  its  charac- 
teristic appearance,  which  has  been  compared  to  a  tennis  racquet. 

The  spores  may  easily  be  stained  by  the  methods  already  described  (p.  145). 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  tetanus  bacillus  is  an  anaerobic  organism 
though  not  so  strictly  anaerobic  as  the  bacillus  of  malignant  oedema.  Growth 
will  take  place  in  media  containing  small  quantities  of  oxygen  and  the  organism 
can  be  trained  to  grow  in  a  slightly  rarefied  atmosphere. 

Growth  takes  place  at  all  temperatures  between  14°  and  43°  C.  Below 
20°  C.  the  growth  is  very  poor.  Spores  form  very  slowly  below  25°  C.  The 
optimum  temperature  is  38°  C.  At  42°  and  43°  C.  the  bacilli  multiply 
rapidly  but  few  of  them  form  spores. 

The  tetanus  bacillus  grows  on  the  ordinary  neutral  or  slightly  acid  or 
alkaline  media  made  from  broth  provided  they  be  made  with  fresh  broth 
(Kitasato).  Ordinary  fresh  beef  broth,  Martin's  broth  or  Nicolle's  medium 
(vide  infra)  are  among  the  best,  while  such  media  as  white  of  egg  and  fresh 
serum  yield  very  poor  growths. 

Debrand  has  shown  that  in  presence  of  the  Bacillus  subtilis  the  tetanus 
bacillus  can  be  easily  cultivated  in  broth  in  contact  with  air.  Under  these 
conditions  the  tetanus  bacillus  retains  its  properties  and  secretes  as  powerful 
a  toxin  as  when  grown  under  strictly  anaerobic  conditions. 

Characters  of  growth.  Broth. — Under  anaerobic  conditions  growth  is 
rapid  at  a  temperature  of  37°  C.  After  incubating  for  about  24  hours  the 
medium  is  generally  cloudy  and  small  bubbles  of  gas  will  be  seen  rising  to 
the  surface  of  the  medium.  The  turbidity  increases  during  the  next  few 
days  and  after  incubating  for  about  a  fortnight  growth  begins  to  slacken 
and  a  precipitate  falls  to  the  bottom  of  the  tube,  the  broth  becoming  clear. 

During  cultivation,  hydrogen,  nitrogen  and  hydrocarbons  are  given  off  in 
moderate  quantity.  The  culture  has  a  characteristic  but  most  disagreeable 
odour,  which  has  been  compared  to  that  of  burnt  hoofs. 

The  tetanus  bacillus  forms  indol  in  broth. 

Gelatin.  Deep  stab  culture. — A  deep  stab  culture  in  a  tube  of  gelatin  from 
which  the  air  has  been  removed  gives,  after  incubating  for  4  or  6  days 
at  a  temperature  of  about  20°  C.,  a  growth  of  small  cloudy-looking 
points  from  which  numerous  very  fine  spicules  shoot  out  at  right  angles  to 
the  line  of  the  stab.  The  cloudiness  extends  and  gradually  invades  the 
whole  of  the  gelatin,  which  commences  to  liquefy  about  the  tenth  day.  A 
flocculent  deposit  forms  at  the  bottom  of  the  tube  and  above  it  the  gelatin 
is  clear  and  liquid.  Spores  only  form  when  the  gelatin  has  begun  to  liquefy. 
Bubbles  of  gas  are  also  formed. 


BIOLOGICAL  PROPERTIES  541 

Single  colonies  in  a  Vignal's  tube. — Small  whitish  points  appear  after  incu- 
bating for  about  4  or  6  days  :  these  soon  form  cloudy  spheres  from  which 
fine  spicules  radiate  on  all  sides.  Bubbles  of  gas  appear  around  the  colonies. 
Liquefaction  of  the  gelatin  commences  about  the  tenth  or  fifteenth  day 
and  progresses  slowly.  The  colonies  appear  as  whitish  flakes  floating  in  the 
liquefied  gelatin. 

Agar. — A  deep  stab  culture  in  agar  incubated  at  37°  C.  rapidly  gives  rise 
to  a  cloudy  growth  which  is  not  very  characteristic.  The  agar  is  split  by 
numerous  gas  bubbles. 

Serum. — In  stab  culture  in  coagulated  serum,  covered  after  sowing  with  a 
layer  of  agar,  a  cloudy  growth  results.  The  serum  is  not  liquefied. 

Potato. — The  tetanus  bacillus  grows  on  potato  under  anaerobic  conditions, 
but  poorly  and  with  difficulty.  In  an  experiment  of  Vaillard  and  Vincent 
the  bacillus  formed  a  thin,  moist,  glistening  layer  rather  like  that  of  the 
typhoid  bacillus  and  microscopically  was  seen  to  be  made  up  of  long  rods 
without  spores. 

Milk.— The  bacillus  grows  in  milk  and  does  not  coagulate  the  medium. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Vitality  and  virulence. 

The  spores  of  the  tetanus  bacillus  are  very  difficult  to  sterilize.  In  a 
closed  vessel  and  in  a  moist  atmosphere  they  withstand  a  temperature  of 
80°  C.  for  6  hours,  90°  C.  for  more  than  2  hours,  and  the  temperature  of 
boiling  water  for  3  or  4  minutes  ;  but  they  can  be  destroyed  with  certainty 
by  boiling  for  8  .minutes. 

Spores  dried  and  mixed  with  soil  and  kept  exposed  to  the  air  but  in  the 
dark,  retain  their  vitality  and  virulence  for  several  months  (Kitasato),  but 
if  dried  on  paper  or  silk  thread  and  exposed  to  air  and  diffused  daylight  or 
direct  sunlight  they  rapidly  undergo  profound  modification. 

These  modifications  vary  with  the  length  of  time  of  exposure.  At  first,  the 
germination  of  the  spores  is  slower  and  their  growth  less  rapid.  Later  they  develop 
into  non-sporing,  non-pathogenic  bacilli  and  finally  perish.  All  these  changes  can 
be  brought  about  in  less  than  a  month.  But  when  exposed  to  light  alone  in  the 
absence  of  air  the  dried  spores  are  more  resistant ;  in  the  experiments  of  Vaillard 
and  Vincent  they  were  still  able  to  germinate  and  give  rise  to  spore- bear  ing  toxi- 
genic  bacilli  after  more  than  2  months,  during  which  they  were  exposed  to  sunlight 
for  59  hours. 

The  tetanus  bacillus  in  the  superficial  layers  of  the  soil  is  evidently  continuously 
exposed  to  these  destructive  and  attenuating  influences,  and  it  rapidly  disappears 
if  by  passage  through  the  alimentary  canal  of  herbivora  it  does  not  find  conditions 
favourable  to  life  and  multiplication  (Sanchez  Toledo  and  Veillon). 

If  dried  in  pus  or  albuminous  fluids  or  on  porous  substances — such  as 
splinters  of  wood  taken  from  tetanus  infected  wounds — the  bacillus  retains 
its  virulence  and  vitality  for  a  long  time. 

2.  Toxin. 

Faber,  and  after  him  Vaillard  and  Vincent,  by  filtering  broth  cultures  of 
the  organism  obtained  a  very  toxic  liquid  the  inoculation  of  which  into 
animals  gave  rise  to  a  typical  attack  of  tetanus. 

Preparation  of  tetanus  toxin. — Experience  has  shown  that  the  composition 
of  the  medium  has  considerable  influence  on  the  toxin  content  of  the  product. 
Peptone -beef-broth  is  the  best  medium  for  preparing  tetanus  toxin. 

Sow  the  bacillus  in  fresh  peptone-beef-broth  and  arrange  the  flask  as 


542  THE   TETANUS   BACILLUS 

described  on  p.  94.  Incubate  at  38°  C.  anaerobically  for  4  or  5  weeks  then 
filter  the  culture  through  a  Chamberland  bougie.  In  this  way  a  very  toxic 
liquid  is  obtained  of  which  a  dose  of  ^5  c.c.  is  fatal  to  a  mouse  when  inocu- 
lated sub-cutaneously. 

Marie  advised  the  addition  of  a  little  gelatin  to  the  broth. 

Ch.  Nicolle  proposes  the  following  medium  : 

Water,         -  -         100      c.c. 

Peptone,     -  2      grams. 

Gelatin  (extra  quality),          ...  1      gram. 

Salt,  -  0-5      „ 

Grow  beneath  a  layer  of  va«eline  oil  (p.  97).  This  medium  yields  a  toxin  in 
6-10  days  which  is  fatal  to  mice  in  doses  of  j^Joo  c*c' 

The  toxicity  of  broth  cultures  may  also  be  increased  to  a  remarkable  degree  by 
making  use  of  the  property  possessed  by  the  organism  of  growing  in  a  medium 
which  has  already  served  for  the  growth  of  the  bacillus  and  in  which  it  has 
elaborated  its  toxin. 

After  incubating  tetanus  bacilli  in  broth  for  3  weeks  filter  the  culture  through 
a  bougie  and  sow  the  filtrate  with  the  bacillus.  Incubate  again  for  3  weeks  and 
filter  a  second  time.  To  the  filtrate,  add  about  TT-th  of  its  volume  of  fresh  sterile 
broth  and  sow  a  third  time  with  the  bacillus.  This  third  culture  when  filtered 
gives  a  very  powerful  toxin. 

Action  of  tetanus  toxin  on  living  animals.— The  inoculation  of  very  small 
doses  of  toxin  is  fatal.  The  most  powerful  toxins  obtained  by  Vaillard  and 
Vincent  by  the  methods  just  described  will  kill  guinea-pigs  in  doses  of  O001  c.c. 
and  mice  in  doses  of  0*00001  c.c. 

If  a  sub-lethal  dose  of  toxin  be  inoculated  a  local  tetanus  results  involving 
only  the  muscles  in  the  neighbourhood  of  the  site  of  inoculation. 

Tetanus  toxin  diffuses  very  quickly  through  the  tissues.  If  a  fraction  of 
a  drop  of  the  toxin  be  inoculated  towards  the  distal  end  of  a  rat's  tail,  in  a 
part,  that  is,  where  absorption  is  very  slow,  the  tail  may  be  cut  off  2  or  3  cm. 
on  the  proximal  side  of  the  site  of  inoculation  three-quarters  of  an  hour 
after  the  operation  without  in  any  way  affecting  the  course  of  the  disease  ; 
the  animal  dies  almost  as  quickly  as  the  control. 

Tetanus  toxin  inoculated  sub-cutaneously  or  intra-muscularly  only  passes 
in  minute  quantities  into  the  blood  but  is  absorbed  by  the  peripheral  expan- 
sions of  the  neurones  and  carried  gradually  to  the  cells  of  the  central  nervous 
system,  producing  in  them  lesions  which  are  responsible  for  the  characteristic 
spasms.  Tetanus  toxin  has  a  special  affinity  for  the  nerve  cells.  This  can 
be  demonstrated  in  vitro. 

Wassermann  and  Takaki,  by  mixing  an  emulsion  of  cerebral  substance  in  normal 
saline  solution  with  tetanus  toxin  and  centrifuging  the  mixture,  obtained  an 
opalescent  solution  containing  almost  no  toxin  at  all.  The  toxin  had  not  been 
destroyed  by  this  treatment  but  was  simply  fixed  by  the  nerve  substance  in  the 
same  way  as  a  dye  might  be  fixed  to  a  fabric.  The  toxin  merely  combines  loosely 
with  the  cerebral  substance  from  which  it  may  be  again  set  free  ;  its  nature  is  not 
altered  (Metchnikoff  and  Marie,  Danysz).  Neutral  mixtures  of  brain  emulsion  and 
tetanus  toxin  become  toxic  on  keeping  :  the  toxin  diffuses  into  the  surrounding  liquid 
and  is  again  free  in  solution.  On  the  other  hand,  toxic  mixtures  of  toxin  and  anti- 
toxin become  in  time  less  toxic  (Knorr).  An  emulsion  of  carmine  in  normal  saline 
solution  acts  in  a  similar  manner :  provided  that  it  has  not  been  sterilized  in  steam 
nor  macerated  it  fixes  the  toxin  and  renders  the  filtrate  harmless  (Stoudensky). 

In  these  mixtures  the  toxin  is  fixed  by  the  particles  of  cerebral  substance  or 
carmine  respectively  and,  if  injected  into  animals,  it  has  no  time  to  diffuse  before 
the  leucocytes  absorb  and  destroy  it. 

In  the  living  tissues,  tetanus  toxin  induces  phenomena  similar  to  those 
just  studied  in  vitro.  Tetanus  toxin  inoculated  sub-cutaneously  into  guinea- 
pigs  is  fixed  after  the  lapse  of  some  hours  by  the  cells  of  the  central  nervous 


TETANUS   TOXIN  543 

system  and  it  is  then  that  the  symptoms  of  tetanus  show  themselves.  Direct 
proof  of  this  fact  is  afforded  by  the  severity  of  the  symptoms  produced  by  the 
inoculation  of  the  toxin  into  the  intact  nervous  tissue.  For  example,  rabbits 
are  very  resistant  to  sub-cutaneous  or  intra-venous  inoculation  of  tetanus 
toxin  ;  to  produce  a  fatal  result  in  4  days  a  dose  of  2*5  c.c.  must  be  used. 
But  a  dose  of  Ol  c.c.  of  the  same  toxin  inoculated  intra-cerebrally  is  fatal 
in  less  than  20  hours.  In  this  case  the  disease  runs  a  special  course — the 
cerebral  tetanus  of  Roux  and  Borrel.  The  animal  exhibits  an  extraordinary 
degree  of  excitability,  is  subject  to  hallucinations,  to  sudden  fears,  and, 
in  short,  to  mania.  Later,  intermittent  convulsive  crises,  motor  troubles 
and  polyuria  appear ;  these  symptoms  terminate  in  the  death  of  the  animal. 
Guinea-pigs  and  rats  suffer  similarly  from  cerebral  tetanus  after  the  inocula- 
tion of  very  small  doses  of  toxin  into  the  brain. 

The  resistance  of  rabbits  to  sub-cutaneous  and  intra-venous  inoculation 
is  not,  therefore,  due  to  a  relative  insusceptibility  of  the  nerve  cells  but  to 
the  fact  that  much  of  the  toxin  injected  does  not  reach  the  cells,  being  destroyed 
(probably  by  the  phagocytes)  in  some  part  of  the  body  as  yet  undetermined 
(Roux  and  Borrel). 

Technique  of  intra-cerebral  injection. — After  incising  the  soft  parts  make  a  hole 
in  the  skull  with  a  gimlet  being  careful  to  protect  the  dura  mater  from  injury : 
then  plunge  the  needle  of  the  syringe  to  the  desired  depth — previously  determined 
by  a  probe — and  inject  the  toxin.  The  animals  stand  these  intra-cerebral  inocula- 
tions very  well  and  it  is  possible  to  inject  8  drops  of  sterile  broth  in  2  stabs  into  a 
guinea-pig's  brain  without  producing  symptoms.  In  a  rabbit  0'5  c.c.  can  be  simi- 
larly inoculated. 

Nature  of  tetanus  toxin. — The  product  of  the  tetanus  bacillus  (or,  tetano- 
spasmin)  is  extraordinarily  toxic. 

Evaporated  in  vacuo,  1  c.c.  of  a  toxin  which  is  fatal  to  mice  in  doses  of  0*00001  c.c. 
gives  a  constant  residue  of  0'04  gram.  On  calcining,  this  residue  loses  0'025  gram 
which  represents  the  organic  matter.  This  organic  matter  consists  largely  of 
inactive  substances  such  as  peptone,  etc.  (Vaillard  and  Vincent) :  but  assuming 
for  the  moment  that  the  whole  of  the  organic  matter  consists  of  toxin  it  follows 
that  these  25  mg.  of  toxin  are  sufficient  to  kill  one  hundred  thousand  mice  ;  on  this 
assumption  the  lethal  dose  of  the  active  principle  for  a  mouse  is  0 '000,000,25  gram. 
And  the  premise  of  the  argument  shows  that  this  is  a  very  conservative  estimate. 

Tetanus  toxin  has  all  the  characteristics  of  enzymes  or  diastases.  Chemi- 
cally, it  is  very  similar  to  diphtheria  toxin.  It  undergoes  considerable 
change  if  heated  to  65°  C.  for  half  an  hour  and  is  completely  destroyed  by 
heating  for  3  hours  at  80°  C.  • 

Stored  in  sealed  vessels  in  the  dark  and  away  from  air,  the  toxin  retains 
its  virulence  for  a  long  time.  The  toxin  is  rapidly  weakened  by  exposure 
to  the  air  and  diffused  light  and  entirely  loses  its  virulence  in  a  few  days 
if  exposed  to  direct  sunlight  and  air. 

Tetanus  toxin  has  the  property  of  adhering  to  amorphous  precipitates 
produced  in  liquids  in  which  it  is  dissolved. 

The  addition  of  calcium  chloride  to  the  toxin  precipitates  calcium  phosphate  and 
a  part  of  the  toxin  is  carried  down  with  the  precipitate.  A  minute  portion  of  this 
precipitate — as  large  as  a  pin's  head — if  carefully  washed  and  inserted  beneath  the 
skin  of  a  guinea-pig,  will  cause  the  death  of  the  animal  from  tetanus  in  30  hours. 
After  precipitation  a  large  amount  of  toxin  still  remains  in  solution  in  the  liquid. 

If  a  little  tetanus  toxin  obtained  by  nitration  be  added  to  a  tube  of  sterile 
gelatin  the  medium  will  be  liquefied  in  a  few  days.  The  phenomenon  is  due 
to  the  presence  in  the  toxin  of  a  peptonizing  diastatic  ferment.  This  ferment 
does  not  appear  to  be  identical  with  the  toxic  principle. 

Evaporated  at  25°  C.  in  vacuv  over  sulphuric  acid  the  toxin  leaves  a  brown, 


544  THE  TETANUS  BACILLUS 

amorphous,  extremely  virulent  residue.  90  per  cent,  alcohol  dissolves  a 
small  quantity  of  this  residue,  and  leaves  after  evaporation  a  whitish,  non- 
toxic  substance  having  a  waxy  smell.  The  remainder  of  the  residue  not 
dissolved  by  alcohol  is  readily  soluble  in  water  and  produces  typical  symptoms 
of  tetanus  in  guinea-pigs  :  it  can  be  precipitated  from  its  aqueous  solution 
by  alcohol.  The  active  substance  contained  in  the  residue  dialyzes  slowly. 

Tetanolysin. — Ehrlich  and  Madsen  have  demonstrated  the  presence  of  an 
haemolysin  in  filtered  cultures  of  the  tetanus  bacillus.  Tetanolysin  differs 
from  tetanus  toxin  :  it  is  highly  unstable  and  is  destroyed  if  heated  for  20 
minutes  at  50°  C.  or  for  some  hours  at  20°  C. 

It  dissolves  the  red  cells  of  domestic  animals  and  especially  those  of  the 
rabbit  and  horse.  Animals  immunized  with  filtered  cultures  rich  in  haemo- 
lysin  elaborate  an  antitetanolysin  simultaneously  with  tetanus  antitoxin. 

Detection  of  toxin  in  the  living  tissues. — When,  in  a  case  of  traumatic 
tetanus,  the  bacillus  cannot  be  found,  an  attempt  may  be  made  to  demon- 
strate the  presence  of  toxin  in  the  blood  by  inoculating  a  few  cubic  centi- 
metres of  the  patient's  serum  into  a  mouse.  The  animal  may  die  but  the 
method  is  not  to  be  relied  upon  and  should  not  in  any  case  be  adopted  when 
the  patient  has  been  treated  with  antitoxin. 

3.  Vaccination. 

(i)  Behring  and  Kitasato  failed  in  their  attempts  to  immunize  animals 
by  repeated  inoculations  of  small  doses  of  tetanus  toxin.  They  obtained 
more  satisfactory  results,  however,  when  they  inoculated  rabbits  with  a 
mixture  of  toxin  and  iodine  terchloride. 

Brieger,  Wassermann  and  Kitasato  by  inoculating  repeated  and  pro- 
gressively increasing  doses  of  cultures  attenuated  by  growing  on  thymus- 
broth  only  obtained  unreliable  results.  Tizzoni  and  Cattani  succeeded  in 
immunizing  rabbits  by  inoculating  them  with  attenuated  cultures. 

Vaillard  was  able  to  immunize  rabbits  with  toxin  which  had  been  partially 
deprived  of  its  toxic  properties  by  heat.  Rabbits  were  inoculated  intra- 
venously on  several  occasions  with  10  c.c.  of  toxin  heated  for  1  hour  first 
at  60°  C.  then  at  55°  C.  and  finally  at  50°  C.  To  increase  the  degree  of 
immunization,  progressively  increasing  doses  of  non-heated  toxin  were  then 
given  viz.  :  first  5  c.c.  then  10  c.c.  followed  by  doses  of  15  and  30  c.c.  Guinea- 
pigs  were  immunized  in  the  same  way. 

(ii)  Roux  and  Vaillard  prefer  to  use,  for  the  purpose  of  vaccination,  toxin 
to  which  a  solution  of  iodine  has  been  added  -(vide  Diphtheria).  Their  toxin 
is  obtained  as  described  above  and  it  should  kill  mice  in  doses  of  0*00025  c.c. 
It  is  mixed  with  Gram's  solution  immediately  before  use.  The  immuniza- 
tion of  a  rabbit  and  of  a  horse  will  be  described  in  illustration  of  the  method. 

Immunization  of  a  rabbit. — Inoculations  given  sub-cutaneously  : — 

1st  day    3  c.c.  of  toxin  mixed  with  1  c.c.  Gram's  solution. 
5th    ;,      5  c.c.  of  toxin +  2  c.c.  of  Gram's  solution. 
9th    „    12  c.c.  of  toxin  +  3  c.c.  of  Gram's  solution. 

On  the  seventeenth  day  the  immunization  of  the  animal  is  complete.  Its  serum 
is  antitoxic.  It  can  then  be  inoculated  at  intervals  of  a  week  with  progressive 
doses  of  5,  10,  15,  20,  30  and  40  c.c.  of  pure  toxin.  Later,  the  inoculations  may 
be  given  intra-venously  or  intra-peritoneally,  and  finally,  a  dose  of  as  much  as 
100  c.c.  of  toxin  can  be  given  at  a  single  inoculation. 

Immunization  of  an  horse. — The  treatment  is  begun  with  a  dose  of  1-5  c.c.  of  a 
mixture  of  equal  parts  of  toxin  and  Gram's  solution  sub-cutaneously.  The  inocula- 
tions are  repeated  every  3  or  4  days.  At  the  end  of  a  fortnight  10  c.c.  of  a  mixture 
of  2  parts  toxin  and  1  part  iodine  solution  are  inoculated  and  the  quantity  is  gradu- 
ally increased  until  about  the  twenty-fifth  day  the  injection  of  pure  toxin  is  com- 


SERUM  THERAPY  545 

menced,  doses  first  of  10,  then  of  15  and  20  c.c.  being  given  every  2  or  3  days.  At 
the  end  of  6  weeks  increasing  doses  of  50,  100,  150  c.c.  can  be  inoculated  into  the 
jugular  vein.  After  these  enormous  doses  intra-venously  the  horse  may  suffer 
from  temporary  disturbances  of  health  such  as  sweating,  colic,  diarrhoea  and  rise 
of  temperature  (1°  or  2°  C.).  Immunization  is  complete  in  about  3  months. 

The  blood  may  be  collected  for  therapeutic  purposes  10  days  after  the  last 
inoculation. 

(iii)  Behring  advises  inoculating  the  horse  in  the  first  instance  with  a 
mixture  of  toxin  and  antitoxin  prepared  in  such  a  way  that  its  inoculation 
into  small  animals  leads  to  a  slight  illness.  The  treatment  is  subsequently 
continued  by  inoculating  pure  toxin.  This  method  is  based  upon  that 
described  for  the  preparation  of  diphtheria  antitoxin  (p.  265). 

(iv)  Vaillard  immunized  a  rabbit  by  injecting  it  sub-cutaneously  on  several 
occasions  with  very  small  doses  of  the  spores  of  tetanus  free  from  toxin  but 
mixed  with  a  little  lactic  acid.  An  animal  treated  in  this  way  resists  the 
inoculation  of  ordinarily  lethal  doses  of  tetanus  toxin  but  its  blood  shows  no 
appreciable  antitoxic  property. 

4.  Serum  therapy. 

The  antitoxic  properties  of  the  blood  of  animals  immunized  against  tetanus 
were  demonstrated  by  Behring  and  Kitasato. 

The  blood  of  an  immunized  rabbit  is  capable  of  neutralizing  tetanus  toxin. 
This  property  is  present  in  serum  free  from  all  cellular  elements  and  is  demon- 
strable both  in  vivo  and  in  vitro.  It  is  not  found  in  the  blood  of  non- 
immunized  animals. 

The  blood  of  naturally  immune  animals,  such  as  the  fowl,  possesses  no  anti- 
toxic property  though  it  easily  acquires  the  property  if  the  animal  be  inoculated 
with  tetanus  toxin.  After  two  or  three  inoculations  of  20  c.c.  each  into  the  peri- 
toneum of  a  fowl  the  blood  of  the  animal  exhibits,  after  the  lapse  of  12—20  days, 
powerful  antitoxic  properties.  Similarly,  the  blood  of  rabbits  immunized  by  the 
inoculation  of  small  doses  of  spores  does  not  possess  antitoxic  properties  but  these 
properties  may  be  conferred  by  inoculating  the  animal  with  tetanus  toxin. 

The  milk  of  immunized  animals  is  also  actively  antitoxic.  The  white  of 
egg  of  fowls  which  have  been  treated  with  toxin  is  not  antitoxic. 

Preparation  of  tetanus  antitoxin. — For  the  practical  application  of  serum 
therapy  to  man  and  animals  the  serum  of  the  horse  is  used.  For  laboratory 
work,  rabbits  are  a  good  source  of  antitoxic  serum. 

Horses  are  immunized  in  the  manner  described  above.1 

After  3  months  the  serum  of  the  horse  is  ready  for  use.  The  antitoxic 
property  is  maintained  and  even  increased  by  inoculating  large  doses  of 
toxin  into  the  jugular  vein  or  beneath  the  skin  of  the  animal  at  intervals. 
After  each  inoculation  the  antitoxic  strength  of  the  serum  is  lowered  for  the 
time  being  ;  the  horse  therefore  must  not  be  bled  until  12  days  have  elapsed. 

The  serum  preserves  its  properties  when  dried  in  vacuo.  In  this  way,  a 
very  powerful  serum  may  be  kept  indefinitely  and  in  small  bulk. 

Standardization  of  the  antitoxin. — In  determining  the  antitoxic  content 
of  a  serum  the  notation  of  Roux  and  Behring  is  used,  which  calculates  the 
content  according  to  the  volume  of  serum  required  to  immunize  1  gram 
weight  of  mouse.  A  serum  is  said  to  be  active  in  10  QQQ  000  if  O'l  c.c.  of  the 
serum  is  sufficient  to  immunize  100  kg.  of  mice  or  when  a  mouse  weighing 
20  grams  is  rendered  immune  by  the  inoculation  of  0'000,002  c.c.  of  serum. 

In  vitro  the  antitoxic  content  of  the  serum  is  measured  by  the  quantity 

1  For  full  details  of  technique,  reference  should  be  made  to  the  section  on  immunization 
against  diphtheria. 

2M 


546  THE  TETANUS  BACILLUS 

of  serum  required  to  neutralize  a  given  volume  of  toxin  of  known  strength. 
The  immunizing  property  of  a  serum  does  not  increase  pari  passu  with  the 
antitoxic  strength. 

Properties  of  tetanus  antitoxin. — In  vitro  the  serum  of  immunized  animals 
mixed  with  tetanus  toxin  neutralizes  the  latter  instantly.  The  volume  of 
serum  required  to  neutralize  a  given  volume  of  toxin  varies  with  the  anti- 
toxic content  of  the  former.  Antitoxic  serums  can  be  obtained  of  which 
0*000,01  c.c.  neutralizes  100  fatal  doses  of  toxin  (Pasteur  Institute,  Paris). 

It  may  be  again  mentioned  that  in  a  toxin- antitoxin  mixture,  the  toxin  is  not 
destroyed  (p.  224) ;  the  toxin  has  merely  entered  into  unstable  combination  with 
the  antitoxin  and  its  toxic  properties  are  easily  brought  into  evidence  again.  Thus, 
for  example,  if  a  guinea-pig  already  inoculated  with  a  culture  of  M.  prodigiosus  or 
B.  coli  be  inoculated  with  a  toxin- antitoxin  mixture  which  is  harmless  for  a  normal 
animal  the  inoculated  animal  soon  shows  symptoms  of  tetanus  intoxication. 

The  inoculation  into  the  peritoneum  of  a  guinea-pig  of  a  dose  of  serum 
(active  in  10  OOQ  QQQ)  equivalent  to  the  three  hundred  and  forty-fifth  part 
of  the  weight  of  the  animal,  rapidly  leads  to  the  manifestation  of  distinct 
antitoxic  properties  in  its  blood.  The  blood  of  a  rabbit  inoculated  with 
Ti(yth  part  of  its  weight  of  serum  is  antitoxic  and  has  marked  immunizing 
properties. 

The  sub-cutaneous  inoculation  of  1  c.c.  of  antitoxic  serum  administered 
10-40  minutes  before  the  inoculation  of  0*0066  c.c.  of  toxin  (a  dose  fatal  to  the 
control  animals  in  48  hours)  protects  guinea-pigs  against  tetanus.  But  in 
animals  inoculated  with  the  toxin  less  than  40  minutes  after  the  inoculation 
of  serum  the  protection  is  not  complete.  Symptoms  of  tetanus  result  the 
severity  of  which  is  inversely  proportional  to  the  length  of  time  elapsing 
between  the  inoculation  of  serum  and  toxin ;  the  longer  the  interval  the 
less  severe  the  symptoms.  But  the  animals  always  recover. 

It  is  a  much  more  difficult  matter  to  prevent  the  onset  of  tetanus  if  serum 
be  given  only  after  the  inoculation  of  toxin,  during,  that  is  to  say,  the  period 
of  incubation.  Similarly,  it  is  less  easy  to  prevent  the  affection  resulting 
from  the  multiplication  of  the  bacillus  in  the  tissues. 

Immunity  is  developed  in  half  an  hour  or  so  after  the  inoculation  of  serum, 
but  it  does  not  last  long,  rarely  exceeding  a  fortnight  or  a  month  in  the  case 
of  laboratory  animals  and  a  week  in  the  case  of  the  human  subject. 

Roux  and  Vaillard  summarize  their  investigations  on  the  prevention  of  tetanus 
as  follows  : 

"  1.  Anti- toxic  serum  even  in  extremely  small  doses  will  certainly  protect  against 
tetanus  if  inoculated  before  the  toxin.  The  immunity  conferred  by  the  serum  is 
transitory.  It  begins  to  dimmish  after  about  a  fortnight  and  disappears  altogether 
in  about  6  or  7  weeks. 

"  2.  When  serum  and  toxin  are  inoculated  at  the  same  time  a  local  tetanus  always 
results  however  large  the  dose  of  serum. 

"  3.  When  the  serum  is  inoculated  after  the  toxin  but  before  the  appearance  of 
any  symptom  of  tetanus  a  local  tetanus  is  always  observed.  The  longer  the  admini- 
stration of  serum  is  delayed  the  larger  must  be  the  quantity  administered.  After 
the  lapse  of  a  certain  length  of  time — varying  in  different  animals — prevention  is 
impossible  even  though  very  large  quantities  of  serum  be  used. 

"  4.  Tetanus  develops  more  or  less  rapidly  and  is  therefore  more  or  less  easy  to 
prevent  according  to  the  site  of  inoculation  of  the  toxin.  (Animals  inoculated  in 
the  paw  are  more  resistant  than  those  inoculated  beneath  the  skin  of  the  thorax 
or  abdomen.) 

"  These  conclusions  refer  to  moderate  doses  of  toxin. 

"5.  When  infection  is  due  to  the  bacilli  multiplying  in  the  tissues,  prevention 
again  depends  upon  the  amount  of  serum  inoculated  and  on  the  time  elapsing  between 
the  moment  of  infection  and  the  administration  of  the  serum.  When  animals  are 


TETANUS  ANTITOXIN  547 

inoculated  in  such  a  way  as  to  suffer  from  an  attack  of  the  disease  which  rapidly 
runs  its  course  the  serum  will  in  most  cases  be  ineffective.  It  may  succeed  in  slowly 
developing  infections  but  in  these  cases  again  the  prevention  of  the  disease  is  not 
always  certain  unless  the  site  of  infection  be  excised.  The  disease  may  appear  to 
be  checked  but  it  is  liable  to  break  out  again  and  terminate  fatally  even  after  a 
considerable  lapse  of  time." 

The  cure  of  the  disease  after  the  symptoms  have  manifested  themselves  is 
therefore  always  difficult  because  the  appearance  of  the  symptoms  is  itself 
evidence  that  the  nerve  elements  are  already  involved.  Antitoxin  neutralizes 
toxin  circulating  in  the  body  but  is  totally  ineffective  against  existing  lesions. 
Very  large  doses  of  the  more  powerful  serums  are  without  effect  on  a  rapid 
case  of  tetanus  :  such  doses  render  the  blood  antitoxic  and  immunizing  but  the 
disease  pursues  its  course.  In  less  severe  cases  the  serum  prolongs  life  but 
unless  the  focus  of  infection  be  removed  the  disease  will  develop  so  soon  as 
the  antitoxic  power  of  the  blood  shall  begin  to  diminish. 

In  man  also,  the  serum  therapy  of  tetanus  has  yielded  but  indifferent 
results.  The  treatment  fails  in  the  more  severe  forms  of  the  disease.  It 
only  seems  to  give  positive  results  in  sub-acute  or  chronic  cases,  and  it  is 
well  known  that  these  cases  when  treated  by  the  ordinary  methods  often 
terminate  in  recovery.  However  that  may  be,  the  serum  treatment  of 
tetanus  is  harmless  and  should  be  adopted  in  all  cases  of  the  disease  in  man. 

Roux  and  Vaillard  define  the  course  of  treatment  in  cases  of  tetanus  in  man  as 
follows : 

"  Inoculate  at  once,  and  without  waiting,  100  c.c.  of  a  very  powerful  serum,  and  if 
possible  excise  the  infected  area.  Another  100  c.c.  should  be  administered  on  the 
following  day  and  on  the  day  after  that.  If  the  symptoms  are  checked,  and  especi- 
ally if  the  focus  of  infection  has  not  been  excised,  give  a  further  dose  of  serum  ten 
days  later  to  obviate  any  recurrence  of  the  disease  such  as  has  been  described  as 
taking  place  in  animals." 

In  view  of  the  failure  of  treatment,  attention  has  been  directed  to  the  pre- 
vention of  the  disease  in  man  and  animals.  Whenever  a  contused  wound 
soiled  with  earth  has  to  be  treated  it  is  well  to  inoculate,  as  a  precautionary 
measure,  20-30  c.c.  of  antitetanus  serum  and  the  inoculations  should  be 
repeated  in  doses  of  10-15  c.c.  every  week  until  all  danger  of  tetanus  has 
vanished.  Nocard,  in  veterinary  practice,  has  obtained  very  good  results 
with  prophylactic  inoculations  in  cases  of  wounds  of  the  foot  and  after 
castration. 

Calmette  has  shown  that  animals  can  easily  be  immunized  against  tetanus  by 
sprinkling  a  small  wound  involving  the  whole  thickness  of  the  dermis  with  dry 
powdered  serum  (vide  ante).  As  a  prophylactic  measure,  he  advises  the  use  of  dried 
serum  for  dressing  wounds  liable  to  be  infected  with  tetanus  and  especially  in  those 
countries  where  the  disease  is  rife. 

Intra-cerebral  inoculation. — These  facts  render  it  difficult  to  understand 
how  tetanus  toxaemia  continues  to  develop  in  animals  which  have  been 
treated  with  antitoxin  and  whose  blood  is  prophylactic  and  antitoxic.  The 
researches  of  Roux  and  Borrel  have  thrown  some  light  on  the  mode  of  action  of 
antitoxin  and  have  given  a  new  impetus  to  the  serum  treatment  of  the  disease. 

A  neutral  mixture  of  toxin  and  antitoxin  is  harmless  to  the  nerve  cells.  It 
can  be  inoculated  into  the  brain  of  a  rabbit  without  any  untoward  incident 
occurring.  Again,  a  rabbit  immunized  with  serum,  while  unaffected  by  the 
sub-cutaneous  inoculation  of  a  quantity  of  toxin  which  is  five  times  the  lethal 
dose  for  an  untreated  animal,  will  nevertheless  die  if  O'l  c.c.  of  toxin  be 
inoculated  into  the  brain.1  However,  its  blood  is  so  antitoxic  that  a  few 

1  This  quantity  when  inoculated  sub-cutaneously  is  absolutely  without  any  effect  on  a 
normal  rabbit. 


548  THE   TETANUS   BACILLUS 

drops  neutralize  considerable  quantities  of  toxin.  And  further,  a  trace  of 
the  blood  accidentally  spilt  in  the  path  of  the  inoculating  needle  as  it 
traverses  the  brain  is  sufficient  to  neutralize  the  toxin  and  the  animal  does 
not  die. 

Antitoxin  given  sub-cutaneously  or  intra-venously  remains  in  the  blood. 
It  has  no  affinity  for  the  nerve  elements.  These  latter,  on  the  other  hand, 
pick  out  and  fix  the  toxin  (vide  ante).  In  an  animal  suffering  from  tetanus 
the  antitoxin  given  sub-cutaneously  or  intra-venously  limits  the  poisoning 
by  destroying  the  toxin  circulating  in  the  blood  but  it  does  not  come  in 
contact  with  the  toxin  already  fixed  by  the  nerve  elements,  which  diffuses 
from  cell  to  cell  and  so  extends  its  ravages.  Antitoxin  should,  therefore, 
not  be  inoculated  into  the  blood  of  those  suffering  from  the  disease  but  rather 
into  the  nerve  centres.  Proof  of  the  correctness  of  these  views  is  afforded 
by  the  results  of  direct  inoculation  of  antitoxin  into  the  brain.1  Roux  and 
Borrel  took  a  number  of  guinea-pigs  and  inoculated  them  sub-cutaneously 
with  a  lethal  dose  of  toxin.  Twenty-four  hours  later  these  animals  showed 
symptoms  of  tetanus.  Some  of  them  were  then  inoculated  with  1  c.c.  of 
serum  sub-cutaneously  and  in  spite  of  this  they  died.  The  remainder  were 
treated  with  4  drops  of  the  same  serum  in  each  cerebral  hemisphere  with  the 
result  that  the  disease  was  arrested  and  the  animals  recovered,  though  some 
of  them  suffered  from  localized  spasms  for  a  long  time.  In  these  cases  the 
inoculation  preserved  the  upper  parts  of  the  cord  against  the  diffusion  of  the 
toxin  but  was  without  effect  on  the  lesions  already  present  in  the  lower  parts  ; 
hence  the  persistence  of  the  spasms  present  at  the  time  of  the  therapeutic 
inoculation.  Similar  experiments  showed  that  when  the  upper  part  of  the 
cord  is  affected  the  inoculation  is  too  late  and  fails  to  save  the  life  of  the 
animal. 

The  cure  of  human  tetanus  would  seem  to  follow  from  these  investigations. 
Unfortunately,  in  practice,  the  results  are  very  inconstant.  Against  the 
rare  cases  of  recovery  following  intra-cerebral  inoculation  of  antitoxin  recorded 
by  Lucas-Championniere,  Girard,  Chauffard,  Nimier,  Ledoux,  Maunoury, 
Holub  and  others  have  to  be  recorded  the  numerous  failures  and  even  rapidly 
fatal  accidents  reported  by  other  observers  (Vallas,  Girard,  Tailhefer  and 
others). 

5.  Agglutination. 

The  serum  of  normal  persons  does  not  agglutinate  the  tetanus  bacillus. 
In  cases  of  tetanus  in  man,  the  blood  does  not  acquire  agglutinating  properties 
(Courmont).  The  same  is  true  of  laboratory  animals. 

Normal  horse  serum  agglutinates  the  tetanus  bacillus  feebly  (1  in  50  to 
1  in  100).  The  serum  of  an  highly  immunized  horse  agglutinates  the  bacillus 
in  dilutions  of  1  in  2,000  and  even  in  1  in  50,000. 

The  inoculation  of  antitetanus  serum  only  confers  an  agglutinating  pro- 
perty when  given  in  considerable  doses  (Courmont). 

SECTION  IV.— DETECTION  AND  ISOLATION  OF  THE  TETANUS 

BACILLUS. 

When  it  is  desired  to  isolate  the  bacillus  from  or  to  detect  its  presence  in 
soil,  a  small  quantity  should  be  inoculated  into  a  guinea-pig ;  the  bacillus 
if  present  can  be  easily  demonstrated  in  and  isolated  from  the  tissues  after 
death. 

1  Inoculation  of  antitoxin  into  the  spinal  cord  is  difficult  to  effect  without  producing 
lesions  and  moreover  does  not  appear  to  be  as  efficient  as  intra-cerebral  inoculation. 
Sub-arachnoid  inoculation  does  not  give  satisfactory  results. 


ISOLATION   OF  THE   TETANUS   BACILLUS  549 

The  examination  of  the  dead  bodies  of  man  or  the  lower  animals  for  the 
tetanus  bacillus  should  be  strictly  limited  to  the  site  of  the  infection. 

It  has  already  been  pointed  out  that  the  bacillus  only  exceptionally  becomes 
generalized  in  the  tissues  of  inoculated  animals.  In  man,  cases  have  been  recorded 
in  which  the  bacillus  has  been  found  in  the  blood  and  also,  but  very  rarely,  in  the 
lymphatic  glands  at  a  distance  from  the  infected  wound. 

The  investigation  should  take  the  following  lines  : 

(a)  Microscopical  examination. — Examine  the  pus  or  membranous  material 
from  the  wound  in  films  stained  with  carbol-crystal-violet  or  dilute  carbol- 
fuchsin  and  by  Gram's  method. 

It  is  often  necessary  to  prepare  several  films  before  the  bacillus  can  be  found,  as 
it  occurs  in  very  small  numbers  and  moreover  its  presence  may  be  masked  by  the 
.large  number  of  other  organisms  present.  Occasionally,  the  reverse  is  the  case  and 
the  tetanus  bacillus  occurs  hi  fairly  large  numbers  while  other  organisms  are  scanty. 
Fig.  259  is  a  reproduction  of  a  film  of  pus  from  a  case  of  this  kind  in  which  the 
presence  of  the  bacillus  was  easily  and  rapidly  demonstrated. 

If  microscopical  examination  fail  to  reveal  the  bacillus  there  is  no  justifica- 
tion for  concluding  that  the  organism  is  not  present ;  cultures  must  then 
be  sown  with  the  material. 

(6)  Cultures.  Isolation. — Kitasato  was  the  first  to  devise  a  means  of 
isolating  the  tetanus  bacillus  in  pure  culture  from  pus  containing  the  organism. 
The  method  is  based  upon  the  resistance  of  the  spore  to  heat  and  upon  the 
anaerobic  properties  of  the  bacillus,  and  is  as  follows  : 

1.  Sow  the  pus  or  other  material  from  the  infected  wound  in  beef  broth 
and  incubate  in  vacuo  at  38°  C. 

2.  After  5  days  the  broth  now  cloudy  contains  together  with  other  anaerobic 
bacteria  numerous  pin-shaped  [drum-stick]  bacilli,  which  are  readily  identified 
as  tetanus  bacilli.     In  order  to  isolate  the  organism,  pipette  a  little  of  the 
impure  culture  into  a  fine  tube,  such  as  the  narrow  part  of  a  Pasteur  pipette, 
seal  the  tube  at  both  ends  in  the  flame  and  heat  at  100°  C.  for  1  or  2  minutes. 
The  spores  of  the  tetanus  bacillus  survive  while  most  of  the  other  organisms 
are  killed. 

3.  Sow  the  contents  of  the  heated  tube  in  broth  and  incubate  in  vacuo  : 
in  the  resulting  growth  the  tetanus  bacillus  predominates  and  may  even 
be  in  pure  culture.     By  thus  alternately  heating  and  cultivating  two  or  three 
times  the  bacillus  can  be  isolated  in  pure  culture. 

4.  Still,  it  often  happens  that  the  tetanus  bacillus  cannot  be  altogether 
freed  from  the  bacillus  of  malignant  oedema  and  another  anaerobic  bacillus 
which  is  not  pathogenic  and  the  spore  of  which  is  not  strictly  terminally 
situated.     In  a  case  such  as  this  the  experiment  will  have  to  be  completed 
by  isolation  in  a  Vignal's  or  Veillon's  tube  (p.  103). 

(c)  Experimental  inoculation. — Inoculate  a  little  of  the  pus  or  a  small 
fragment  from  the  infected  wound  directly  into  guinea-pigs  or  mice.  Symp- 
toms of  tetanus  soon  follow  the  inoculation.  Cultures  also  should  be  tested 
by  inoculation  into  animals. 

Bacillus  botulinus.1 

The  Bacillus  botulinus  was  isolated  by  van  Ermengem  from  some  ham  the 
consumption  of  which  at  Ellezelles  in  December  1895  was  followed  by  the 
symptoms  of  botulism  2  in  a  number  of  persons  who  had  partaken  of  it  and 
of  whom  three  died. 

Homer  investigated  a  similar  epidemic  at  Alsfeld  and  isolated  an  organism  prac- 
tically identical  with  van  Ermengem' s  bacillus. 

1  This  section  has  been  added.  2  Botulus,  a  sausage. 


550  BACILLUS  BOTULINUS 

Fischer  and  Landmann  have  also  recorded  an  outbreak  of  food  poisoning  at 
Darmstadt  attributable  to  the  same  organism.  In  this  epidemic  the  illness  was 
traced  to  some  preserved  peas  mixed  with  which  were  some  fragments  of  meat. 
Twenty- one  persons  were  affected  of  whom  eleven  died. 

Van  Ermengem  recovered  the  bacillus  from  the  spleen  of  a  patient  who  had  died 
of  botulism.  Kempner  isolated  the  Bacillus  botulinus  from  the  excreta  of  pigs. 

The  Bacillus  botulinus  is  now  generally  accepted  to  be  the  specific  cause  of 
botulism.  It  appears  to  be  normally  a  saprophyte  which  does  not  multiply  to  any 
extent  in  the  living  body.  Botulism  is  regarded  therefore  as  a  toxaemia  due  to 
the  consumption  of  meat  which  has  been  accidentally  contaminated  with  the  B. 
botulinus  and  in  which  the  toxin  is  present  at  the  time  it  is  eaten. 

The  toxin  is  quite  easily  destroyed  by  boiling  and  by  other  methods  of  cooking 
so  that  the  danger  of  botulism  is  limited  to  the  consumption  of  uncooked  or  very 
insufficiently  cooked  food  ;  and  Sacquepee  points  out  that  the  symptoms  of  botulism 
only  follow  the  consumption  of  "  preserved  "  foods,  that  is  to  say,  of  such  articles 
of  diet  as  sausages  which  are  made  some  months  before  they  are  eaten,  ham,  meats 
preserved  in  tins  and  bottles,  game  pies,  etc. 

Experimental  inoculation. — The  inoculation  of  a  large  quantity  (10-20  c.c.) 
of  an  emulsion  of  a  culture  of  the  bacillus  or  of  an  infected  food  proves  fatal 
to  rabbits  in  48  hours.  Small  doses  lead  to  a  chronic  illness  which  however 
ultimately  terminates  in  the  death  of  the  inoculated  animal. 

Guinea-pigs  and  mice  are  also  susceptible. 

The  most  characteristic  results  are  obtained  with  cats.  *  The  inoculation 
of  cultures  of  the  Bacillus  botulinus  into  cats  is  followed  by  a  typical  attack 
of  botulism  with  its  characteristic  symptoms  of  bulbar  paralysis. 

Morphology. — The  Bacillus  botulinus  is  a  straight,  rod-shaped,  slightly 
motile  organism  with  rounded  ends.  It  measures  from  4^-9/x  x  0'9-1'2/A.  The 
bacilli  are  often  seen  in  pairs  and  in  short  chains  and  resemble  closely  the 
bacilli  of  malignant  oedema  and  quarter  ill. 

Under  favourable  conditions,  e.g.  on  an  alkaline  gelatin  medium  incubated 
at  20°-25°  C.  the  bacillus  forms  oval  spores  ;  these  are  generally  terminally 
situated  and  are  rather  wider  than  the  bacillus.  Spores  are  not  formed  at 
37°  C. 

The  Bacillus  botulinus  stains  with  the  ordinary  aniline  dyes  and  is  gram- 
positive  though  relatively  somewhat  easily  decolourized. 

Cultural  characteristics. — The  bacillus  is  a  strictly  anaerobic  organism. 
The  optimum  temperature  of  growth  is  below  that  of  the  majority  of  patho- 
genic micro-organisms,  viz.  20°-30°  C. 

The  bacillus  will  not  grow  except  on  media  made  with  meat,  and  pig's  flesh  is 
better  than  that  of  either  cattle  or  sheep. 

In  agar  media  it  produces  a  considerable  amount  of  gas  with  a  strong 
odour  of  butyric  acid. 

In  gelatin  the  growth  is  characteristic.  Circular,  transparent,  yellowish- 
brown  colonies  appear  in  4-6  days  and  around  each  colony  the  medium  is 
liquefied.  The  gas  which  is  formed  either  splits  the  medium  or,  if  the  latter 
be  largely  liquefied,  rises  to  the  surface  and  forms  a  layer  of  bubbles. — In 
stab  culture  the  growth  assumes  an  arborescent  form  (Distaso). 

In  milk  the  bacillus  grows  slowly  but  does  not  coagulate  the  medium 
(Distaso).  Von  Hibler  however  finds  that  the  B.  botulinus  first  coagulates 
the  milk  and  then  peptonizes  the  clot. 

Vitality. — The  spores  of  the  Bacillus  botulinus  retain  their  vitality  for  about 
a  year.  They  can  be  destroyed  in  15  minutes  at  85°  C.  and  in  30  minutes 
at  80°  C. 

Toxin. — Van  Ermengem  was  the  first  to  show  that  the  Bacillus  botulinus 
produces  an  extra-cellular  toxin  which  has  all  the  generic  properties  of  the 


BACILLUS   BOTULINUS  551 

extra-cellular  toxins  of  diphtheria  and  tetanus.  Its  action  is  specific  and  it 
always  gives  rise  to  symptoms  of  botulism ;  moreover  the  illness  which 
follows  its  administration  is  always  preceded  by  an  incubation  period. 

Preparation. — The  toxin  may  be  prepared  by  any  of  the  methods  described  for 
the  preparation  of  tetanus  toxin. 

Properties. — The  toxicity  of  the  toxin  is  lowered  by  exposure  to  light  and  air 
and  by  heating  at  58°  C.  for  3  hours.  Its  properties  are  destroyed  altogether  by 
heating  at  a  temperature  of  100°  C. 

The  toxin  is  highly  poisonous.  For  man  the  lethal  dose  is  said  to  be  0*035  mg.  ; 
rabbits  succumb  in  72  hours  and  guinea-pigs  in  4-5  days  after  being  inoculated 
with  quantities  of  0'005  c.c.  ;  in  cats  a  dose  of  0'5  c.c.  of  a  filtered  culture  produces 
the  characteristic  symptoms  of  botulism  and  death  takes  place  in  about  8-10  days. 

The  toxin  acts  specifically  on  the  nervous  system. 

Kempner  and  Pollack,  and  Marinesco  have  demonstrated  changes  of  toxic  degenera- 
tion in  the  anterior  columns  of  the  cord.  Kempner  and  Schepilewski  have  shown 
that  the  toxin  combines  with  the  tissues  of  the  brain  and  spinal  cord  and  that  an 
emulsion  of  these  tissues  has  the  property  of  neutralizing  the  toxin — cf.  Wasser- 
mann  and  Takaki's  experiments  on  tetanus  toxin  (p.  542). 

Charrin  and  Bardier  have  shown  that  the  toxin  acts  also  specifically  on  the  heart. 

Antitoxin. — Kempner  by  adopting  the  ordinary  procedure  (vide  Diphtheria 
and  Tetanus)  has  been  able  to  prepare  an  antitoxic  goat  serum.  Unfortunately 
it  would  appear  that  to  obtain  any  curative  effect  in  animals  the  antitoxin 
must  be  administered  within  12  hours  of  the  administration  of  the  toxin  so 
that  it  seems  probable  that  the  use  of  the  antitoxin  in  medical  practice  will 
not  yield  any  very  striking  results. 


CHAPTER  XXXVII. 

THE  BACILLUS  OF  QUARTER  ILL. 
(BACILLUS  CHAUVJEI.) 

Introduction. 

Section  I. — The  experimental  disease,  p.  552. 

1.  Susceptible  animals,  p.  552.     2.  Methods  of  infection,  p.  553.     3.  Symptoms 
and  lesions,  p.  553. 
Section  II. — Morphology,  p.  554. 

1.  Microscopical  examination  and  staining  reactions,  p.  554.     2.  Cultural  charac- 
teristics, p.  555. 
Section  III. — Biological  properties,  p.  556. 

1.  Vitality  and  virulence,  p.   556.     2.  Vaccination,  p.   556.     3.  Toxin,  p.   558. 
4.  Serum  therapy,  p.  559.     5.  Agglutination,  p.  560. 

THE  bacillus  of  quarter  ill,  symptomatic  anthrax  or  black  quarter  was  dis- 
covered by  Arloing,  Cornevin  and  Thomas. 

Quarter  ill  is  a  disease  of  cattle  and  also,  but  rarely,  of  sheep  and  goats  ;  other 
animals  are  not  subject  to  the  spontaneously  contracted  disease.  Cattle  are  only 
affected  between  the  ages  of  4  months  and  5  years  ;  in  the  early  weeks  of  life  they  are 
immune  and  again  become  insusceptible  after  5  years  of  age. 

The  virus  is  present  in  soil  and  infection  takes  place  through  the  skin,  trachea 
and  probably  the  alimentary  canal  (Arloing).  Epizootics  occur  generally  in  the 
summer  and  are  particularly  prevalent  in  certain  districts  :  for  instance  in  the 
Pyrenees,  in  the  district  of  Haute  Marne,  and  in  Switzerland.  The  disease  is  almost 
always  fatal  and  is  responsible  for  a  heavy  mortality  among  cattle. 

Quarter  ill  has  the  clinical  symptoms  of  a  septicaemia :  the  temperature  is  raised, 
the  animal  is  dull,  loses  its  appetite  and  ceases  to  chew  the  cud :  swellings  appear 
on  the  limbs,  in  the  angle  of  the  jaws,  in  the  throat,  on  the  thorax  and  in  the  testicles  ; 
the  swellings  most  commonly  involve  the  neighbouring  muscles,  where  they  rapidly 
grow  to  a  very  large  size  becoming  emphysematous  and  crepitant  in  the  centre  ; 
the  oedema  increases  peripherally  and  the  animal  dies  in  3-5  days. 

SECTION  I.— THE  EXPERIMENTAL  DISEASE. 
1.  Susceptible  animals. 

Guinea-pigs,  oxen  and  sheep  are  very  susceptible  to  experimental  inocula- 
tion :  goats  less  so.  Donkeys  and  horses  suffer  from  a  painful  oedematous 
swelling  at  the  site  of  (sub-cutaneous)  inoculation  but  rapidly  recover. 

Rabbits  are  immune,  but  their  immunity  can  easily  be  overcome  (Roger, 
Leclainche  and  Vallee). 

If  the  site  of  inoculation  be  traumatized  or  if  a  little  lactic  acid  or  a  small  quantity 
of  a  culture  of  Micrococcus  prodigiosus  be  inoculated  with  the  bacillus  the  resistance 


EXPERIMENTAL  INOCULATION  553 

of  the  tissues  is  broken  down  and  the  phagocytic  action  of  the  leucocytes  hindered, 
with  the  result  that  a  septicsemic  condition  develops.  Leclainche  and  Vallee 
obtained  in  this  way  cultures  very  virulent  for  the  species,  3  c.c.  of  which  sub- 
cutaneously  injected  was  sufficient  to  cause  death. 

Mice,  rats,  dogs,  pigs,  cats  and  birds  are  immune.1 

2.  Methods  of  infection. 

The  Bacillus  chauvcei  being  a  strict  anaerobe  only  grows  when  inoculated, 
deeply  into  the  tissues,  and  does  not  infect  superficial  wounds. 

The  influence  of  the  site  of  inoculation  is  very  marked.  The  same  dose 
of  the  virus  which  will  kill  an  ox  when  inoculated  into  the  cellular  tissues  of 
the  body  will  set  up  merely  a  benign  infection  if  inoculated  into  the  con- 
nective tissue  of  the  neck. 

In  the  latter  case  however  if  the  inoculated  part  be  warmed  the  bacillus 
will  multiply  and  the  animal  may  die. 

The  influence  of  temperature  is  emphasized  by  the  fact  that  frogs  under  normal 
conditions  resist  infection  but  succumb  to  the  disease  if  kept  in  the  incubator  at  25°  C. 
after  being  inoculated. 

The  inoculation  of  a  virulent  virus  into  the  veins  of  an  ox  merely  leads  to 
a  temporary  rise  of  temperature  :  but  if  at  the  time  of  inoculation  a  trace 
of  the  virus  gain  access  to  the  peri  vascular  tissues  a  fatal  septicaemia  ensues. 
Infection  may  also  be  established  by  rupturing  a  blood  vessel  after  intra- 
venous inoculation  (Arloing). 

The  disease  may  be  produced  experimentally  in  several  ways  : 

(i)  By  the  inoculation  of  cultures. — If  ordinary  cultures  be  inoculated  the 
result  will  be  uncertain,  but  if  young  cultures  in  Martin's  broth  be  used 
infection  will  certainly  follow  :  three  or  four  drops  inoculated  beneath  the 
skin  will  kill  a  guinea-pig  (500  grams)  in  18-24  hours  (Leclainche  and  Vallee). 

(ii)  By  inoculation  of  infected  blood. — Blood  from  the  heart  of  a  guinea-pig 
or  sheep  recently  dead  of  the  disease  should  be  sealed  up  in  pipettes,  incubated 
for  48  hours  and  then  used  for  inoculation. 

(iii)  By  inoculation  of  serous  exudates.  Method  recommended. — The  fluid 
from  the  local  oedemata  may  be  inoculated  without  heating  :  take  a  portion 
of  one  of  the  swellings  and  crush  it  up  in  a  mortar  with  a  little  sterile  water 
and  inoculate  the  emulsion.  The  dried  exudate  (Arloing's  powder,  vide 
infra]  may  also  be  used  :  in  this  case  grind  up  a  little  of  the  powder  with  a 
few  drops  of  sterile  water  and  add  a  trace  of  lactic  acid. 

(iv)  By  inoculation  of  spores  alone. — By  applying  methods  similar  to  those 
described  in  the  chapter  on  malignant  oedema  (Chap.  XXXVIII.),  Leclainche 
and  Vallee  have  shown  that  the  inoculation  of  spores  free  from  toxin  does  not 
lead  to  the  death  of  the  animal :  but  the  spores,  like  those  of  the  bacillus  of 
malignant  oedema,  will,  if  protected  from  the  action  of  the  phagocytes, 
germinate  and  originate  a  fatal  disease  ;  certain  substances  and  certain 
organisms  (e.g.  Staphylococcus  aureus)  will  also  assist  the  germination  of  the 
spores  and  so  favour  infection. 

3.  Symptoms  and  lesions. 

The  disease  produced  experimentally  in  the  guinea-pig  will  be  taken  as 
the  type. 

If  the  material  be  inoculated  into  the  muscles  of  the  thigh  a  painful  and 

1  It  has  been  suggested  that  the  bacillus  of  malignant  oedema  and  the  bacillus  of  quarter 
ill  are  identical  species.  This  position  however  cannot  be  maintained,  for  apart  from 
morphological  and  cultural  differences,  they  differ  widely  in  their  pathogenicity ;  all 
the  animals  which  have  just  been  mentioned  as  immune  to  the  latter  bacillus  are  suscep- 
tible to  the  bacillus  of  malignant  oedema. 


554  THE   BACILLUS   OF   QUARTER  ILL 

characteristic  swelling  soon  appears  surrounded  by  an  cedematous  infiltration 
which  rapidly  encroaches  on  the  abdominal  wall.  The  animal  curls  itself 
up  and  does  not  move  :  its  coat  is  dull  and  rough  and  the  hair  can  be  easily 
pulled  out  from  over  the  oedematous  area  :  death  takes  place  2  or  3  days 
after  inoculation. 

Post  mortem. — The  characteristic  appearances  are  limited  to  the  local 
lesion.  The  site  of  inoculation  is  marked  by  a  swelling,  and  the  neighbouring 

connective  tissue  is  infiltrated  with  an  exudate 
rich  in  red  cells  ;  the  muscles  are  yellowish  or 
dull  red  in  colour  while  the  fibres  are  vitreous 

"  and  degenerated.     The  centre  of  the  swelling 

is  black  and  sanious  and  contains  bubbles  of 

^  gas.     The  lesions  have  a  peculiar  odour,  which 

Arloing  compared  to  that  of  rancid  butter,  and 

.  ^  A         contain  numerous  bacilli.     The  area  of  oedema 

f  ^  extends   for   a  variable   distance  around  the 

swelling  and  invades  the  abdominal  and  thoracic 

f  walls,  and  the  exudate  is  rich  in  bacilli  but 

,-,  contains  no  leucocytes.     The  inguinal   glands 

FIG.  262.-The  bacillus  of  quarter    are  .^ematous  and  swollen.     In  the  peritoneal 

ill.    Scraping  from  muscle  —  Dilute    cavity  there  is  a  small  quantity  of  a  slightly 

Rerff)fuchsin'     (0c-  n'  obj-  Ath,    cloudy  exudate  containing  numerous  bacilli  and 

the    intestine    is    frequently    congested.     The 

blood  is  almost  unaltered  in  appearance  :  during  life  bacilli  cannot  be  found 
on  microscopical  examination  :  after  death  they  are  present  though  few  in 
number  even  after  incubation  at  37°  C. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance  and  staining  reactions. 

The  bacillus  of  quarter  ill  is  a  small  rod-shaped  organism  (3-8/*  x  1/u)  and 
occurs  singly  or  in  pairs  :  it  is  straight  and  rigid  and  has  absolutely  square- 
cut  ends.  The  bacillus  is  motile  but  the  motility  can  only  be  observed  under 
anaerobic  conditions  in  the  centre  of  the  microscopical  preparations. 

The  bacillus  of  malignant  oedema  differs  from  the  bacillus  of  quarter  ill 
in  the  following  respects.  It  forms  long  chains,  which  are  never  met  with 
either  in  cultures  or  tissues  in  the  case  of  the  latter  bacillus  ;  it  is  also  more 
slender,  more  wavy,  longer  and  more  motile,  than  the  bacillus  of  quarter  ill. 

Spores  are  rapidly  formed  in  the  muscular  swellings  but  may  not  appear 
if  death  occur  very  soon.  The  spore  is  first  apparent  as  an  oval  refractile 
spot  in  the  centre  or  towards  the  end  of  the  bacillus  often  giving  rise  to  an 
appearance  like  a  tennis  racquet  or  clock  pendulum.  As  a  rule  spores  are 
not  found  in  the  fluid  which  accumulates  about  the  swellings  and  are  never 
seen  in  serous  exudates  :  in  cultures,  both  bacilli  and  spores  are  formed. 

The  relatively  large  size  of  the  bacillus  renders  the  examination  of  unstained 
preparations  easy. 

Staining  reactions.— The  bacillus  of  quarter  ill  stains  readily  with  solutions 
of  the  basic  aniline  dyes  containing  a  mordant.  It  is  gram-positive  and 
retains  the  stain  by  Claudius'  method. 

The  spores  may  be  stained  in  the  usual  way  (p.  146).  They  do  not  stain 
in  the  cold  with  aqueous  solutions  of  the  aniline  dyes. 

(a)  Cultures.— For  cultures  the  most  useful  stains  are  carbol-crystal-violet 
or  dilute  carbol-fuchsin. 

(b)  Smear  preparations. — Films  may  be  prepared  from  scrapings  from  the 


MORPHOLOGY  555 

swellings,  by  smearing  a  little  of  the  peritoneal  exudate  or  oedema  fluid  on  a 
cover-glass,  or  by  rubbing  a  cover-glass  lightly  over  the  surface  of  the  liver 
of  a  guinea-pig  dead  of  the  disease.  These  films  may  be  stained  with  carbol- 
crystal-violet,  Gram's  stain  or  Claudius'  stain. 

(c)  Sections. — The  swellings  should  be  used  for  cutting  sections  and  are 
best  stained  with  Kuhne's  carbol-blue  or  by  Gram's  method  with  counterstain. 

Note. — The  tissues  of  carcases  dead  of  symptomatic  anthrax  rapidly  become 
invaded  by  the  bacillus  of  malignant  oedema,  a  normal  inhabitant  of  the  intestine. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  bacillus  of  quarter  ill  is  a  strict  anaerobe 
and  must  therefore  be  cultivated  by  the  methods  applicable  to  anaerobic 
organisms. 

The  media  should  be  recently  prepared.  Ordinary  broth  is  not  a  good 
medium  and  though  the  addition  of  glycerin  (4  per  cent.),  glucose  (1  per 
cent.),  or  sulphate  of  iron  (1  per  cent.),  etc.  has  been  proposed,  such  additions 
are  said  to  offer  no  advantages  (Kitasato).  Leclainche  and  Vallee  obtained 
very  good  results  with  recently  prepared  Martin's  broth.  A  considerable 
amount  of  gas  is  formed  in  culture  (H,  C02,  CH4)  which  has  a  characteristic 
odour  of  butyric  acid. 

Growth  commences  at  15°  C.  the  optimum  temperature  being  about  37°  C. 

For  isolating  the  organism  the  peritoneal  exudate  or  heart  blood  or  some  of  the 
fluid  from  the  swelling  should  be  used  for  sowing  cultures,  but  it  is  rather  difficult 
to  obtain  pure  cultures :  in  the  pulp  from  the  swellings  the  bacillus  is  almost 
always  associated  with  other  organisms  (facultative  aerobes,  Vibrion  septique,  etc,). 

It  is  necessary  also  to  test  the  purity  of  the  culture  from  time  to  time  by  micro- 
scopical examination  (as  indicated  above,  the  appearance  of  long  and  wavy  threads 
denotes  a  contamination  with  the  Vibrion  septique)  and  by  the  inoculation  of  a 
rabbit  and  guinea-pig.  If  only  the  guinea-pig  die  the  virus  is  pure  and  four  or  five 
drops  of  blood  from  the  heart  should  be  sown  immediately  after  death — this  affords 
in  any  case  a  certain  method  of  obtaining  pure  cultures. 

Maintenance  of  virulence. — In  the  laboratory  the  virulence  of  the  micro-organism 
is  maintained  by  passage  through  guinea-pigs.  It  should  be  remembered  that 
infection  with  the  Bacillus  chauvcei  predisposes  in  a  marked  degree  to  infection 
with  the  Vibrion  septique  and  that  after  a  few  passages  the  animals  die  of  a  double 
infection. 

Culture  media.  Broth. — Recently  prepared  Martin's  broth  is  better  than 
ordinary  broth.  After  incubating  for  about  20 
hours  at  37°  C.  there  is  a  general  cloudiness  of 
the  medium  and  gas  is  given  off  in  considerable 
amount.  After  2  or  3  days  flocculi  form  which  fall 
to  the  bottom  and  the  broth  gradually  becomes 
clear. 

Albuminous  media. — The  growth  is  more  luxur- 
iant and  the  virulence  is  maintained  for  a  longer 
time  than  in  broth.  Serum  or  serum  diluted 
with  two  parts  of  sterile  water  or  meat  juice 
prepared  according  to  the  method  given  when 
dealing  with  the  Vibrion  septique  should  be  utilized. 

Gelatin.  Deep  stab  cultures. — Small  irregularly  FIG.  263.— The  bacillus  of  quar- 
spherical  colonies  throwing  out  lateral  offshoots  ^tkIffie™^OTa*UC(^ 
slowly  make  their  appearance  along  the  line  of  the 

needle  puncture  when  the  medium  is  incubated  at  20°  C.  :  when  they  begin 
to  become  confluent  the  gelatin  is  split  by  the  formation  of  gas  bubbles.  The 
culture  spreads  irregularly  and  liquefies  the  medium. 


556  THE   BACILLUS   OF   QUARTER   ILL 

Discrete  colonies. — Small  whitish  spheres  with  lateral  offshoots  appear  in 
the  depth  of  the  gelatin  which  subsequently  become  cloudy  and  irregular 
in  shape.  Gas  is  formed  and  the  gelatin  liquefied. 

Agar.  Deep  stab  cultures. — When  incubated  at  37°  C.  a  cloudy  whitish 
line  rapidly  appears  along  the  line  of  the  stab,  the  agar  is  then  broken  up 
by  the  gas  evolved  and  the  gaps  are  invaded  by  the  culture. 

Potato. — No  apparent  growth. 

Milk. — Abundant  growth. 

White  of  egg.— White  of  egg  is  not  sensibly  attacked  ( Jungano  and  Distaso). 

SECTION  HI.— BIOLOGICAL  PROPERTIES. 
1.  Vitality  and  virulence. 

The  bacillus  of  quarter  ill  has  considerable  vitality  and  since  it  forms 
spores  is  able  to  resist  the  ordinary  methods  employed  for  the  destruction  of 
micro-organisms.  In  cultures,  the  spores  resist  a  temperature  of  100°  C.  for 
several  minutes.  If  some  of  the  exudate  in  the  muscles  be  dried  the  spores 
can  only  be  killed  by  exposure  to  moist  heat  at  110°  C.  for  several  hours. 
Ordinary  antiseptic  solutions  and  putrefactive  changes  have  no  action  on 
the  spores. 

The  virulence  of  the  organism  disappears  somewhat  rapidly  in  culture  : 
after  being  sub-cultivated  five  or  six  times  in  broth  it  fails  to  set  up  a  fatal 
disease  in  guinea-pigs.  Its  virulence  in  culture  is  readily  destroyed  by  physical 
agents,  e.g.  an  exposure  to  a  temperature  of  100°  C.  for  2  minutes  renders  it 
a  virulent :  serum  cultures  are  more  resistant  than  broth  cultures. 

In  the  dried  exudate  from  swellings  on  the  other  hand  the  virulence  is  as 
persistent  as  the  vitality ;  in  such  an  exudate  the  bacilli  after  being  dried 
at  35°  C.  will  remain  alive  and  virulent  for  years.  They  are  able  also  to 
resist  exposure  to  high  temperatures  for  several  hours  (see  under  Vaccina- 
tion, infra). 

In  the  living  tissues  the  organism  retains  its  properties  for  a  long  time :  e.g.  if 
a  frog  which  has  been  inoculated  but  has  resisted  infection  be  left  for  a  fortnight 
or  3  weeks  and  then  be  put  in  the  incubator  at  25°  C.  it  will  suffer  from  the  disease 
(Arloing). 

2.  Vaccination. 

One  attack  of  symptomatic  anthrax  confers  immunity,  and  the  disease 
does  not  recur  in  the  same  subject. 

1.  The  injection  of  the  virus  into  the  veins  of  an  ox,  a  harmless  proceeding 
if  properly  done,  will  render  the  animal  immune  (Arloing,  Cornevin,  and 
Thomas).  Cattle  can  be  vaccinated  by  inoculating  5  or  6  c.c.  of  a  virulent 
exudate  into  the  jugular  vein  but  on  account  of  inherent  difficulties  and 
dangers  it  has  been  abandoned  as  a  practical  method  of  vaccination. 

(ii)  The  principle  of  a  method  of  vaccination  devised  by  Arloing,  Cornevin 
and  Thomas  is  the  inoculation  of  an  attenuated  virus  into  the  tip  of  the  tail. 
It  has  already  been  mentioned  that  inoculation  in  this  situation  is  not  a 
severe  method  of  infection.  The  attenuated  virus  is  prepared  by  exposing 
dried  serous  exudate  from -the  muscles  to  the  action  of  heat  as  follows  : 

The  infected  muscles  of  an  ox  or  sheep  dead  of  quarter  ill  are  finely  minced  and 
added  to  two-thirds  their  weight  of  sterile  water.  The  mixture  is  rubbed  up  in  a 
mortar  and  filtered  through  muslin :  the  filtrate  is  poured  on  to  porcelain  plates 
and  kept  in  a  dry  incubator  at  35°  C.  until  completely  dry,  after  which  the  dried 
extract  is  ground  up  in  a  pepper  mill  and  then  in  a  mortar  until  reduced  to  a  very 
fine  powder.  In  this  way  a  very  virulent  product  is  obtained  which  retains  its 
toxicity  for  years. 


VACCINATION  557 

To  attenuate  the  product  some  of  the  powder  is  mixed  with  twice  its  weight  of 
sterile  water,  spread  in  a  thin  layer  and  heated  at  100°  C.  or  105°  C.  for  5-6 
hours.  This  constitutes  the  first  vaccine  and  on  inoculation  leads  to  no  untoward 
symptoms. 

Another  portion  of  the  powder  is  similarly  moistened  and  heated  for  5  or  6 
hours  at  90°  or  94°  C.  only.  This  is  the  second  vaccine  ;  it  is  more  virulent  than 
the  first  and  is  dangerous  to  inoculate  in  the  first  instance. 

To  vaccinate  an  animal  emulsify  in  a  mortar  the  contents  of  a  packet  of 
the  first  vaccine  with  10  c.c.  of  boiled  water,  stirring  meanwhile.  Filter  the 
emulsion  through  a  piece  of  fine  linen  and  inoculate  1  c.c.  of  the  filtrate  into 
the  tip  of  the  tail  (or  into  the  tip  of  the  ear)  after  cutting  the  hair  and  washing 
the  skin  with  soap  and  water.  Some  days  later  the  immunity  is  strengthened 
by  inoculating  the  second  vaccine. 

The  immunity  is  permanent  and  accidents  are  uncommon.  If  the  reaction 
be  over-violent,  which  is  not  the  case  if  the  inoculation  has  been  properly 
done,  the  animal's  tail  should  be  cut  to  remove  the  focus  of  infection.  The 
method  is  now  extensively  practised  in  France  and  Switzerland. 

The  full  virulence  of  the  powder  can  be  restored  if  necessary  by  mixing  a  little 
lactic  acid  with  it ;  the  acid  produces  a  small  necrotic  focus  and  prevents  the  leuco- 
cytes reaching  the  site  of  inoculation,  and  so  allows  the  organism  to  develop. 
Alcohol,  trauma  and  certain  micro- organic  toxins  act  in  the  same  way.  The  organism 
in  the  powders  is  never  really  attenuated  :  it  is  true  that  the  virus  is  modified  but 
only  in  the  sense  that  its  germination  is  retarded,  during  which  time  the  inoculated 
tissues  have  time  to  arrange  their  phagocytic  defences.  Proof  of  this  view  is 
furnished  by  the  fact  that  when  a  little  of  the  powder  is  sown  on  artificial  media  a 
virulent  culture  is  obtained. 

Arloing's  powders  contain  numerous  impurities  since  contaminating  micro- 
organisms are  present  in  very  large  numbers  in  the  swelling.  It  is  to  these 
latter  that  the  occasional  accidents  which  occur  during  vaccination  are  to 
be  attributed.  Leclainche  and  Vallee  have  described  a  method  by  which 
vaccines  containing  pure  cultures  of  the  organism  may  be  prepared  : 

The  heart  blood  of  a  guinea-pig  or  sheep  dead  of  quarter  ill  is  collected  and  incu- 
bated in  sealed  capsules  for  48  hours  at  37°  C.  The  contents  are  then  spread  in 
thin  layers  in  sterile  Petri  dishes  and  kept  in  the  incubator  at  37°  C.  until  desiccation 
is  complete.  The  dried  blood  is  powdered  and  rubbed  up  with  a  little  sterile  water. 
This  paste  is  spread  in  thin  layers  on  glass  plates  and  divided  into  two  portions. 
One  is  heated  in  an  hot  air  chamber  for  7  hours  at  a  temperature  of  102°  C.  (first 
vaccine),  the  other  at  a  temperature  of  92°  C.  (second  vaccine).  The  vaccines  are 
powdered  and  stored  in  sterile  tubes  in  the  same  way  as  Arloing's. 

Note. — Pure  vaccines  behave  like  spores  of  the  bacillus  in  that  they  are  not  mixed 
with  toxin  and  are  not  fatal  to  animals  in  moderate  doses,  but  on  the  other  hand 
they  are  efficient  for  purposes  of  vaccination  which  is  not  the  case  with  pure  spores 
(obtained  by  heating  cultures  to  80°  C.  for  3  hours).  If  spores  be  inoculated  they  are 
phagocyted  immediately,  whereas  the  physical  state  of  the  vaccine  when  inoculated 
to  a  certain  extent  retards  phagocytosis  so  that  the  destruction  of  the  spores  is 
delayed  until  the  tissues  have  had  time  to  elaborate  antibodies,  which  antibodies 
constitute  the  immunity  of  the  animal. 

(iii)  Kitasato  and  also  Kitt  state  that  the  inoculation  of  broth  cultures 
more  than  a  fortnight  old  does  not  kill  guinea-pigs  but  confers  immunity 
upon  them,  and  that  virulent  cultures  heated  at  80°  C.  for  30  minutes  have 
similar  vaccinating  properties  whereas  if  heated  at  80°  C.  for  3  hours  these 
properties  are  destroyed. 

(iv)  Leclainche  and  Vallee  have  shown  that  virulent  cultures  heated  at 
70°  C.  for  2  hours  do  not  kill  young  guinea-pigs  but  are  toxic  for  adult  guinea- 
pigs.  One  c.c.  of  a  culture  treated  in  this  way  confers  a  lasting  immunity 
on  young  guinea-pigs  :  2  c.c.  inoculated  into  cattle  behind  the  shoulder 


558  THE   BACILLUS   OF   QUARTER  ILL 

gives  rise  to  trifling  symptoms  and  confers  a  certain  degree  of  immunity  ;  a 
second  inoculation  7  days  later  completes  the  immunization. 

On  these  facts  Leclainche  and  Vallee  have  based  the  following  method  of 
vaccination. 

Pure  cultures  are  sown  in  Martin's  broth  and  after  incubating  for  5—8  days  are 
distributed  in  glass  ampoules,  which  are  then  sealed  and  heated  in  a  water  bath  at 
70°  C.  :  this  is  the  first  vaccine.  The  second  vaccine  is  prepared  in  a  similar  manner 
but  the  ampoules  are  not  heated.  This  method  does  away  with  all  need  for  powdering 
the  vaccines  and  yields  a  vaccine  of  which  the  dosage  is  simple. 

Vaccination  with  these  pure  vaccines  is  hot  unattended  by  danger,  and  in  a  later 
paper  Leclainche  and  Vallee  recommend  the  administration  of  a  dose  of  immunizing 
serum  as  a  condition  precedent  to  the  inoculation  of  the  pure  attenuated  virus 
(vide  infra). 

Note. — Animals  vaccinated  against  quarter  ill  with  pure  vaccines  are  not 
immune  to  malignant  oedema  (Leclainche  and  Vallee). 

Roux  and  Chamberland  however  had  stated  that  guinea-pigs  rendered  immune 
to  quarter  ill  are  often  immune  to  the  bacillus  of  malignant  oedema,  and  Diinsch- 
mann  having  obtained  an  antiserum  for  quarter  ill  affirmed  that  it  neutralized 
fatal  doses  of  the  bacillus  of  malignant  oedema.  These  results  were  due  to  the  fact 
that  the  cultures  used  by  the  authors  for  preparing  the  immunizing  vaccines  were 
impure ;  they  immunized  the  annuals  against  both  infections  at  one  and  the  same 
time. 

3.  Toxin. 

(i)  Roux  showed  that  broth  cultures  sterilized  by  filtration  through  porce- 
lain or  by  heating  at  115°  C.  contain  slightly  toxic  soluble  products  which  on 
inoculation  into  animals  produce  a  certain  degree  of  immunity.  He  was 
able  to  immunize  guinea-pigs  by  inoculating  them  with  a  total  quantity  of 
40  c.c.  of  the  sterilized  cultures  administered  on  three  separate  occasions 
at  intervals  of  2  days. 

(ii)  Diinschmann  obtained  a  toxin  which  killed  guinea-pigs  when  given 
intra-peritoneally  in  doses  of  2  c.c.  The  bacillus  was  grown  in  meat  pulp 
(p.  566)  for  7  days ;  the  fluid  was  then  expressed,  filtered  through  porcelain 
and  inspissated  in  vacua  over  sulphuric  acid. 

(iii)  Leclainche  and  Vallee  have  shown  that  the  Bacillus  chauvcei  when 
grown  in  Martin's  broth  produces  a  powerful  toxin  the  inoculation  of  which 
is  not  followed  by  any  incubation  period.  The  toxicity  reaches  its  maxi- 
mum about  the  fifth  day  of  incubation  and  then  diminishes.  The  decanted 
fluid  is  so  rapidly  fatal  to  laboratory  animals  that  the  bacillus  has  not  time 
to  develop.  Three  or  four  drops  of  such  a  culture  inoculated  into  the 
cerebral  hemispheres  of  a  guinea-pig  lead  to  a  fatal  result  in  a  few  hours  :  a 
dose  of  2-5  c.c.  inoculated  into  the  ear  vein  of  a  rabbit  kills  the  animal  in 
from  5  minutes  to  5  hours  :  10  c.c.  injected  into  the  jugular  vein  of  a  horse 
is  fatal  in  about  6  minutes. 

The  toxicity  diminishes  in  presence  of  air :  free  aeration  destroys  the  toxin  in 
48  hours.  The  toxin  is  not  completely  destroyed  by  heating  at  115°  C.,  but  by 
heating  at  70°  or  75°  C.  its  chemiotactic  properties  which  before  were  negative 
become  positive.  A  large  proportion  of  the  toxin  is  held  back  on  filtration,  but  the 
filtrate  is  still  fatal  to  experimental  animals  :  guinea-pigs  die  a  few  hours  after 
the  inoculation  of  5  c.c.  of  the  filtrate  into  the  peritoneal  cavity,  and  in  smaller  doses 
symptoms  of  intoxication  appear  in  7-9  days  and  the  animals  die  from  cachexia. 

Eisenberg  has  confirmed  Leclainche  and  Vallee's  experiments.  A  six-day- 
old  culture  in  Martin's  broth  containing  normal  rabbit  serum,  after  being 
decanted  and  centrifuged,  killed  rabbits  in  a  few  minutes  when  1  c.c.  was 
inoculated  into  a  vein.  This  toxin  is  almost  destroyed  by  heating  at  60°  C. 
and  is  neutralized  by  the  serum  of  a  vaccinated  rabbit. 


SERUM  THERAPY  559 

(iv)  Grassberger  and  Schattenfroh  find  that  the  toxins  produced  by  different 
strains  of  the  Bacillus  chauvcei  are  not  all  of  the  same  degree  of  toxicity,  and 
that  in  order  to  prepare  a  satisfactory  toxin  it  is  necessary  to  select  a  strain 
carefully  and  adapt  it  to  a  medium  suitable  for  toxin  production.  These 
investigators  recommended  a  lactose-broth  containing  calcium  carbonate  as 
the  best  medium  on  which  to  grow  strains  which  ferment  rapidly,  and  broth 
containing  calcium  lactate  for  strains  which  do  not  set  up  a  violent  fermenta- 
tion— since  the  toxicity  is  materially  diminished  by  filtration  (Leclainche  and 
Vallee)  it  is  necessary  to  work  with  decanted  cultures.  In  this  way  Grass- 
berger and  Schattenfroh  obtain  a  toxin  which  is  fatal  to  guinea-pigs  in  2-4 
days  in  doses  of  O'Ol  c.c.  inoculated  sub-cutaneously  (normal  toxin).  Rabbits, 
monkeys,  dogs,  mice,  fowls  and  pigeons  are  also  susceptible  to  the  toxin  : 
inoculation  of  40  c.c.  beneath  the  skin  of  a  calf  is  fatal  in  2-6  days  :  sheep 
succumb  to  the  inoculation  of  2  c.c. 

4.  Serum  therapy. 

(i)  Kitt  was  the  first  to  make  experiments  on  the  serum  therapy  of  symp- 
tomatic anthrax  ;  working  with  sheep  and  horses  he  inoculated  the  virus 
first  intra-venously  and  then  sub-cutaneously.  The  serum  of  these  animals, 
in  doses  of  5-10  c.c.  sub-cutaneously,  protects  sheep  against  a  virulent 
inoculation  given  3  days  to  a  week  later. 

(ii)  Dlinschmann  increases  the  natural  immunity  of  the  rabbit  by  the 
inoculation  of  increasing  doses  of  the  virus.  The  serum  of  the  rabbit  is 
then  both  prophylactic  and  antitoxic  for  the  guinea-pig,  if  given  either 
separately,  before  or  at  the  same  time  as  the  virus,  or  if  well  mixed  with  the 
latter.  The  serum  however  has  no  therapeutic  property. 

(iii)  Arloing  immunized  an  heifer  by  inoculating  it  with  increasing  doses 
of  the  virus  over  a  period  of  6  months'.  The  animal  was  then  immune  to  the 
inoculation  into  the  blood  and  connective  tissues  of  very  large  quantities 
of  fluid  from  a  local  lesion.  Its  blood  was  found  to  possess  prophylactic, 
therapeutic  and  antitoxic  properties. 

Arloing' s  serum  neutralizes  double  its  weight  of  fresh  virulent  virus  in  vitro. 
The  mixture  may  be  safely  inoculated  into  sheep. 

If  inoculated  in  doses  of  10  c.c.  into  the  connective  tissues  of  another  part  of  the 
body  it  will,  if  administered  at  the  same  time,  protect  a  sheep  weighing  30  kg.  against 
a  fatal  dose  of  fresh  virus.  A  similar  result  is  obtained  if  one-tenth  the  dose  be 
inoculated  intra-venously. 

The  immunity  conferred  is  of  short  duration  :  it  is  still  in  evidence  on  the  fourth 
day  but  has  completely  disappeared  at  the  end  of  a  week.  If  however  the  inocula- 
tion of  the  serum  be  followed  by  an  ordinarily  fatal  dose  of  fresh  virus  a  much  more 
stable  immunity  is  established. 

The  inoculation  of  the  mixture  of  serum  and  virus  produces  few  symptoms  but  does 
not  lead  to  any  appreciable  degree  of  immunity.  Animals  treated  with  the  serum- 
virus  mixture  survive  the  test  inoculation  rather  longer  than  control  animals  but 
always  succumb  in  the  end. 

The  sub -cutaneous  inoculation  of  a  powerfully  prophylactic  serum  arrests  the 
extension  of  a  fatal  inoculation  in  sheep,  if  given  within  3  hours  of  the  latter.  The 
same  dose  is  effective  if  given  intra-venously  9  hours  after  infection,  but  has  no 
effect  after  12  hours. 

The  properties  seem  to  be  preserved  intact  if  the  serum  be  rapidly  dried  in  the  air 
in  a  thin  layer  at  a  temperature  of  38°  C. 

(iv)  Leclainche  and  Vallee  obtain  an  antiserum  by  hyper-immunizing 
goats  and  horses. 

(a)  Goats. — First  a  virulent  culture  is  inoculated  into  the  veins,  and  then  at 
intervals  of  10  days,  5,  10,  and  15  c.c.  of  a  filtered  product  obtained  by  crushing 


560  THE   BACILLUS   OF   QUARTER   ILL 

the  muscles  of  infected  guinea-pigs  in  an  equal  volume  of  water  is  inoculated  sub- 
cutaneously. 

The  serum  of  the  goat  after  the  second  sub-cutaneous  inoculation  protects  guinea- 
pigs  against  the  inoculation  of  a  virulent  maceration  provided  that  the  test  inocula- 
tion (0'5  c.c.)  be  made  1-3  days  after  that  of  the  serum  (1'5  c.c.). 

A  mixture  of  the  serum  with  the  virus  is  harmless  :  serum  inoculated  at  the  same 
time  as  the  test  inoculation  but  into  a  different  part  of  the  body  does  not  protect 
the  animal,  and  similarly,  the  serum  is  without  effect  if  inoculated  after  the  virus. 

(6)  Horses. — Horses  are  inoculated  intra-venously  with  10-135  c.c.  of  cultures 
in  Martin's  broth.  The  serum  in  doses  of  1-5  c.c.  immunizes  guinea-pigs  against 
the  inoculation  of  a  drop  of  virulent  exudate.  No  immunity  is  apparent  until  12 
hours  after  the  inoculation  of  the  serum  and  is  of  short  duration — at  the  most  a 
week. 

A  mixture  consisting  of  3  c.c.  of  serum  with  5  drops  of  culture  is  harmless. 
Guinea-pigs  treated  with  such  a  mixture  show  merely  a  transitory  immunity  lasting 
at  the  outside  10  days.  The  serum  is  without  any  therapeutic  effect  on  guinea- 


(v)  Grassberger  and  Schattenfroh  were  unable  to  immunize  guinea-pigs 
with  their  toxin  (supra).  Rabbits  and  cattle  on  the  other  hand  were  easily 
immunized  :  calves  which  had  received  a  total  quantity  of  60—70  c.c.  of 
toxin  in  two  or  three  doses  no  longer  reacted  to  the  inoculation  of  10  c.c.  : 
in  4-5  months  their  serum  was  so  antitoxic  that  0*0025  c.c.  neutralized  1  c.c. 
of  normal  toxin. 

This  serum  is  prophylactic  for  guinea-pigs  if  inoculated  before  the  toxin.  If 
mixed  with  the  toxin  the  mixture  is  neutral  (guinea-pigs,  bovine  animals,  sheep 
and  rabbits)  and  induces  a  permanent  immunity  against  the  toxin  (rabbits,  sheep 
and  cattle,  but  not  guinea-pigs). 

Guinea-pigs  which  have  been  treated  with  a  prophylactic  dose  of  the  antitoxic 
serum  are  immune  to  an  inoculation  of  the  Bacillus  chauvcei.  In  cattle,  prophylactic 
inoculations  of  toxin  or  of  the  toxin -serum  mixture  do  not  always  immunize  against 
the  experimental  disease  but  appear  to  yield  better  results  in  the  case  of  the  naturally 
contracted  infection. 

5.  Agglutination. 

The  serums  of  Leclainche  and  Vallee  agglutinate  the  bacillus  in  dilutions 
of  1  in  30  to  1  in  6,000.  The  serum  of  a  cow  infected  with  quarter  ill  agglu- 
tinates the  bacillus  in  a  dilution  of  about  1  in  300. 

The  serum  of  healthy  animals  only  agglutinates  the  Bacillus  chauvcei  in 
dilutions  of  less  than  1  in  12. 

The  bacillus  of  malignant  oedema  is  not  agglutinated  by  the  serum  in 
dilutions  above  1  in  12  and  in  the  same  way  the  anti-malignant-oedema 
serum  has  no  agglutinating  action  on  the  bacillus  of  quarter  ill :  both 
serums  have  a  strictly  specific  action  (Leclainche  and  Vallee). 


CHAPTER  XXXVIII. 
BACILLUS  MALIGNI  (EDEMATIS. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  561. 

1.  Susceptible  animals,  p.  561.     2.  Methods  of  infection,  p.  562.     3.  Symptoms 
and  lesions  in  experimental  animals,  p.  563. 
Section  II. — Morphology,  p.  563. 

1.  Microscopical  appearance  and  staining  reactions,  p.  563.     2.  Cultural  charac- 
teristics, p.  564. 
Section  III. — Biological  properties,  p.  565. 

1.  Vitality  and  virulence,  p.   565.     2.  Toxin,   p.   565.     3.  Vaccination,  p.   567. 
4.  Serum  therapy,  p.  568.     5.  Agglutination,  p.  568. 

THE  bacillus  of  malignant  oedema  (Fr.  Vibrion  septique)  is  the  oldest  known 
anaerobic  pathogenic  micro-organism.  In  1887  Pasteur  determined  its 
morphology  and  biological  properties  and  described,  as  "  acute  experimental 
septicaemia,"  the  disease  which  follows  the  introduction  of  the  organism  into 
the  sub-cutaneous  cellular  tissue  of  laboratory  animals.  Chauveau  and 
Arloing  showed  that  the  bacillus  of  malignant  oedema  is  the  ordinary  cause 
of  the  rapid  gaseous  gangrene  of  man  *  (gangrenous  septicaemia).  Krannhals 
attributed  "  rag-pickers'  disease  "  to  this  organism.  German  writers  describe 
the  organism  under  the  name  "  Malignes  Odem." 

Certain  traumatic  gangrenes  of  domestic  animals  are  also  caused  by  the 
bacillus  of  malignant  oedema. 

The  bacillus  of  malignant  oedema  is  very  widely  distributed  outside  the 
body.  In  the  spore  form  it  is  found  in  garden  soil,  in  dirt  from  the  street, 
in  the  mud  of  different  waters,  etc.  It  occurs  as  an  harmless  saprophyte 
in  the  intestine  and  in  the  excreta  of  man  and  animals.  After  death  the 
bacillus  may  pass  from  the  intestine  into  the  blood  stream  :  this  infection 
of  the  blood  stream  takes  place  very  rapidly  in  animals  which  have  died  of 
anthrax. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 
1.  Susceptible  animals. 

Most  animals  are  susceptible  to  infection  with  the  bacillus  of  malignant 
oedema  and  guinea-pigs  and  mice  particularly  so  :  one-millionth  of  a  drop 
of  an  infected  exudate  is  sufficient  to  kill  a  guinea-pig  (Davaine). 

1  The  bacillus  of  malignant  oedema  is  not  the  only  cause  of  gangrene.  In  Chap.  XXXIX. 
a  number  of  other  anaerobic  organisms  which  may  be  concerned  in  gangrenous  conditions 
are  described. 

2N 


562  THE   BACILLUS   OF   MALIGNANT  (EDEMA 

Besson  has  shown  that  passage  through  guinea-pigs  increases  the  virulence  of 
the  organism.  A  bacillus  is  rapidly  obtained  of  such  virulence  that  less  than  one- 
hundredth  of  a  drop  of  a  broth  culture  is  fatal  to  guinea-pigs  and  rabbits  in  8  hours, 
and  one  drop  is  fatal  to  cats  in  12—15  hours. 

Babbits  and  white  rats  come  next  in  the  scale  of  susceptibility.  Sheep, 
goats  and  horses  are  also  very  susceptible  ;  the  same  is  true  of  cats,  a  species 
which  is  often  erroneously  said  to  be  only  slightly  susceptible.  Asses,  small 
birds,  fowls  and  pigeons  are  less  susceptible,  next  come  dogs  and  finally  oxen. 

Sewer  rats  are  almost  immune  and  only  succumb  to  considerable  doses  of 
very  virulent  viruses. 

2.  Methods  of  infection. 

The  bacillus  of  malignant  oedema  is  a  strict  anaerobe  and  only  multiplies 
in  the  living  tissues  when  introduced  deeply  beneath  the  skin  or  into  the 
muscles  or  peritoneal  cavity.  It  does  not  lead  to  septicaemia  when  inoculated 
into  the  veins  and  does  not  infect  superficial  wounds  (Chauveau  and  Arloing). 
The  disease  may  be  produced  experimentally  in  animals  in  many  ways. 

(i)  By  inoculation  of  a  culture  or  infected  exudate. — Sub-cutaneous  inocula- 
tion is  a  very  severe  method  of  infection.  Doses  of  less  than  O'Ol  c.c.  will 
rapidly  kill  susceptible  animals. 

(ii)  By  inoculation  of  the  spores  alone.  Ancillary  organisms.— Besson  has 
shown  that  when  the  spores  of  the  bacillus  alone  are  inoculated  into  the  sub- 
cutaneous tissues  of  guinea-pigs  and  rabbits  even  in  considerable  doses 
(4  or  5  million  in  the  case  of  the  guinea-pig,  14  million  in  the  case  of  the 
rabbit),  they  do  not  germinate  but  are  rapidly  phagocyted,  and  the  animal 
shows  no  lesion  other  than  a  small  hard  nodule  at  the  site  of  inoculation 
which  disappears  in  a  few  days. 

Pure  spores  are  easily  obtained  by  destroying  the  toxin  in  broth  cultures  by  heat. 
Aspirate  a  few  drops  of  a  spore- bearing  broth  culture  into  a  small  glass  tube,  seal  it 
at  both  ends  and  keep  it  in  a  water  bath  at  80°  C.  for  3  hours.  If  the  heated  culture 
be  inoculated  even  in  large  quantities  the  animal  suffers  no  ill- effects  but  if  it  be 
sown  in  a  fresh  tube  of  broth  a  very  virulent  culture  is  obtained.  An  even  more 
simple  method  consists  in  using  cultures  which  have  been  in  the  warm  incubator 
(37°  C.)  for  several  months;  under  these  conditions  the  toxin  disappears  and  the 
spores  alone  remain. 

It  is,  however,  only  necessary  to  add  a  small  quantity  of  some  negatively 
chemiotactic  substance  to  spores  to  prevent  the  phagocytes  fulfilling  their 
protective  role  to  induce  a  condition  of  septicaemia.  Thus,  if  a  small 
drop  of  lactic  acid  be  mixed  with  the  spores  and  the  mixture  be  inoculated 
the  animal  will  die.  A  similar  result  is  obtained  if  a  small  quantity  of  the 
toxin  of  the  bacillus,  which  possesses  chemiotactic  properties,  be  added  to 
spores  :  or  if  the  latter  be  mechanically  protected  against  the  action  of  the 
phagocytes — as  by  enclosing  them  in  a  little  piece  of  sterile  filter-paper  or  in  a 
small  cube  of  agar  before  introducing  them  beneath  the  skin  of  a  guinea-pig. 

The  septicaemia  can  be  still  more  easily  produced  by  spores  free  from 
toxin  if  they  be  mixed  with  some  other  micro-organism,  harmless  in  itself 
but  the  secretions  of  which  are  negatively  chemiotactic.  Many  organisms 
found  in  soil  can  be  used  in  this  experiment  as  well  as  other  species  e.g.  the 
Micrococcus  prodigiosus  and  the  Staphylococcus  pyogenes  aureus. 

In  the  same  way,  traumatic  injuries  leading  to  death  of  the  tissues  (burns, 
ligatures,  etc.)  lead  to  a  diminished  activity  on  the  part  of  the  phagocytes 
and  so  favour  the  development  of  the  spores. 

(iii)  By  inoculation  of  soil  containing  spores. — The  inoculation  of  a  trace 
of  mud  from  the  street  or  of  garden  soil  sub-cutaneously  beneath  the  skin  of 
a  guinea-pig  or  rabbit  often  results  in  a  fatal  septicaemia.  The  development 


MORPHOLOGY  563 

of  the  spores  which  are  in  the  soil  is  facilitated  by  the  numerous  other 
organisms  present. 

3.   Symptoms  and  lesions. 

The  septicaemia  of  Pasteur  runs  a  similar  course  in  all  animals  but  the 
duration  of  the  disease  varies  with  the  species  inoculated. 

The  symptoms  in  the  guinea-pig  which  are  typical  of  those  seen  in  other 
animals  are  as  follows  :  very  soon  after  the  inoculation  of  a  trace  of  a  viru- 
lent culture  beneath  the  skin  of  the  thigh  or 
abdomen  an  oedema  forms  at  the  site  of  in- 
oculation  ;  a  few  hours  later  the  animal  with 
its  coat  staring  is  found  crouching  in  a  corner 
of  its  cage  showing  no  inclination  to  move, 
convulsions  soon  appear  and  death  supervenes 
often  in  less  than  12  hours. 

When  the  virulence  of  the  organism  is  very 
high,  the  oedema  is  negligible  and  the  septi 
caemia   develops   very   rapidly,    death    taking 
place  after  a  very  few  hours'  illness. 

Post  mortem,  there  is  more  or  less  oedema 
at  the  site  of  inoculation   the  muscles  in  the      ^  ^  _The  of  nt 

neighbourhood  are  bright  red  and  infiltrated  oedema.  Smear  preparation  from  the 
\Kr\^n  Q  eorrma  ovnrlafn  -«/hilo  f>io  nrmnoo+iTra  surface  of  the  liver  of  a  guinea-pig. 

with  a  serous  exudate,  wmie  tne  connective  Carbol.blue.  (Oc.n.obj.^th, Reich) 
tissue  is  distended  with  bubbles  of  fetid  gas 

and  crepitates  beneath  the  finger.  A  variable  amount  of  almost  clear  serous 
exudate  is  present  in  the  peritoneal  cavity  :  the  liver  is  discoloured  and  the 
spleen  diffluent  while  the  lungs  are  normal  in  appearance.  A  most  disagreeable 
smell  emanates  from  the  carcase. 

The  fluid  of  the  local  oedema  contains  large  numbers  of  bacilli  but  no 
leucocytes.  The  peritoneal  exudate  examined  under  the  microscope  also 
gives  the  appearance  of  a  pure  culture  of  the  bacillus  with  numerous  fila- 
mentous forms  :  spores  are  not  found  in  the  living  animal,  but  are  formed 
rapidly  after  death,  especially  if  the  carcase  be  kept  in  the  incubator  at  a 
temperature  of  35°  C. 

The  bacillus  of  malignant  oedema  is  very  seldom  found  in  the  blood  of 
animals  during  life,  but  it  enters  the  blood  stream  soon  after  death.  If  the 
body  be  left  in  the  incubator  (35°  C.)  for  a  few  hours  and  blood  films  be 
then  made  a  large  number  of  bacilli  will  be  found. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance  and  staining  reactions. 

The  bacillus  of  malignant  oedema  is  a  rod-shaped  organism  measuring 
3-15/x  x  O6-1/A,  more  slender  than  the  anthrax  bacillus,  and  occurring  singly 
or  in  chains.  Chains  are  particularly  numerous  in  the  blood  of  carcases 
kept  for  a  few  hours  at  37°  C.  :  under  these  conditions  the  bacilli  form  long 
filaments  (40/x)  made  up  of  unequal  segments.  The  rods  though  sometimes 
straight  are  more  often  curved  and  wavy ;  their  ends  are  clean  cut  and  very 
slightly  rounded  at  the  angles,  in  contrast  to  the  ends  of  the  anthrax  bacillus 
which  are  sinuous  but  form  a  right  angle  with  the  lateral  surface. 

The  bacillus  is  motile,  but  only  under  anaerobic  conditions,  so  that  the 
centre  and  not  the  edge  of  the  preparation  should  be  examined.  The  vibrios 
move  with  a  slow  undulating  creeping  movement  due  to  flagella  arranged 
laterally  on  the  organism. 


564  THE   BACILLUS   OF  MALIGNANT  (EDEMA 

Spores  are  rapidly  formed  in  the  bodies  of  dead  animals  and  in  cultures. 
The  spore  appears  as  a  brilliant  refractile  oval  point  which  causes  a  swelling 
in  the  centre  or  towards  the  end,  but  very  rarely  exactly  at  the  end  of  the 
bacillus. 

There  appear  to  be  several  varieties  of  the  bacillus  distinguished  by  differences 
of  motility,  degrees  of  virulence  and  the  rapidity  with  which  they  liquefy  serum. 
Great  uncertainty,  however,  still  exists  on  this  point  and  it  is  reasonable  to  suppose 
that  the  so-called  different  varieties  are  merely  modified  forms  of  one  and  the  same 
organism. 

Staining  reactions. — The  bacillus  of  malignant  oedema  is  easily  stained 
by  the  basic  aniline  dyes.  It  is  gram-positive  but  the  reaction  is  inconstant 


FIG.  265.— The  bacillus  of  malignant  FIG.  266.— The  bacillus  of  malignant 
oedema.     Film  from  a  3  days'  growth  oedema.     Flagella.      x  1200. 

on  agar.     Dilute  carbol-fuchsin.    (Oc. 
IV,  obj.  ^th,  Reich.) 

unless  certain  precautions  be   observed.     The   best  dye  is  carbol-gentian- 
violet,  and  it  should  be  left  on  the  film  for  5  minutes  before  being  replaced 
by  the  iodine  solution.     The  bacillus  stains  well  by  Claudius'  method. 
Spores  and  flagella  may  be  stained  by  the  methods  described  in  Chap.  IX. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  bacillus  of  malignant  oedema  is  a  strict  anaerobe 
and  can  only  be  grown  by  the  methods  described  in  Chap.  VI. 

The  bacillus  of  malignant  oedema  is  one  of  the  least  strictly  anaerobic  of  all  the 
anaerobic  organisms  and  grows  quite  well  in  media  from  which  the  air  has  been 
only  partially  removed.  Rosenthal  has  been  able  to  adapt  it  to  aerobic  conditions. 

Growth  does  not  take  place  below  15°  C.  The  optimum  temperature  is 
about  37°  C.  Very  satisfactory  cultures  can  be  obtained  by  incubating  at 
41°  C.  (Besson). 

Culture  media.  Broth. — A  well  marked  cloudiness  appears  after  incubating 
at  37°  C.  for  12  or  20  hours.  Indol  and  evil  smelling  gases  (C00,  H,  hydro- 
carbons and  volatile  sulphur  compounds)  are  produced  in  considerable 
amount.  The  broth  soon  clears,  the  growth  precipitating  to  the  bottom  of 
the  tube.  As  long  as  the  broth  is  cloudy  it  contains  numerous  bacilli  which 
form  spores  after  about  20-24  hours.  In  the  deposit  spores  and  granular 
disintegrated  bacilli  only  are  to  be  found. 

Albuminous  media. — The  culture  is  similar  to  that  in  broth  but  more 
abundant.  Broth  containing  blood  or  ascitic  fluid,  Martin's  broth,  serum 
either  pure  or  diluted  with  an  equal  volume  of  water  or  broth,  and  meat 
juice  sterilized  by  filtration  through  a  Chamberland  bougie  all  give  very 
abundant  growths. 

The  following  medium  appears  to  give  the  best  cultivations  (Besson). — To  500 
grams  of  finely  minced  lean  meat  add  500  c.c.  of  distilled  water  and  a  large  pinch 


BIOLOGICAL   PROPERTIES  565 

of  salt.  Leave  the  mixture  in  the  ice-chest  for  12  or  20  hours.  Decant  the  liquid 
and  squeeze  the  residue  in  a  meat  press.  Add  normal  soda  solution  until  the  reac- 
tion of  the  fluid  is  feebly  alkaline  then  heat  to  115°  C.  for  5  minutes.  Filter  through 
Chardin  paper  and  sterilize  the  dark  brown  filtrate  at  112°  C.  for  20  minutes.  A 
slight  coagulum  forms  during  sterilization  but  dissolves  during  the  growth  of  the 
organism. 

Gelatin.  Deep  stab  culture. — At  20°  C.  growth  begins  after  incubating 
about  48  hours.  Small  cloudy  spheres  appear  along  the  line  of  the  stab  and 
these  rapidly  become  confluent  forming  a  long,  whitish  streak.  Bubbles  of 
gas  then  appear  which  split  the  gelatin  and  the  culture  spreads  irregularly 
along  these  fissures.  Liquefaction  rapidly  follows  and  involves  the  whole 
of  the  medium. 

Single  colonies. — On  the  second  or  third  day  small  cloudy  whitish  spots 
with  ill-defined  margins  appear  in  the  depth  of  the  medium.  The  medium 
liquefies  around  them  and  bubbles  of  gas  are. formed. 

Agar.  Deep  stab  culture. — Growth  in  the  form  of  a  cloudy  whitish  streak 
along  the  line  of  the  stab  very  quickly  appears  on  incubating  at  37°  C.  The 
agar  is  soon  split  by  bubbles  of  gas  and  the  culture  spreads  along  the  fissures. 

Potato. — No  apparent  growth. 

Coagulated  serum. — The  bacillus  grows  and  liquefies  the  serum.  Cooked 
white  of  egg  is  also  digested  by  the  bacillus. 

SECTION   III.— BIOLOGICAL  PROPERTIES. 
1.   Vitality  and  virulence. 

Vitality. — Bacilli  which  are  not  in  the  spore  condition  are  soon  killed  by 
exposure  to  air  or  by  heating  for  a  few  moments  at  60°  C. 

Spores  are  only  formed  in  the  complete  absence  of  air  but  once  formed 
they  are  highly  resistant  to  the  action  of  oxygen.  Ordinary  antiseptic 
solutions  are  almost  without  action  upon  them  (Chauveau  and  Arloing). 
In  the  moist  state  they  resist  temperatures  of  80°  C.  for  several  hours  and 
90°  C.  for  more  than  30  minutes  (Besson).  They  are  only  killed  by  moist 
heat  above  100°  C.  when  contained  in  dried  albuminous  material.  According 
to  Sanfelice  they  are  not  affected  by  exposure  to  sunlight  for  50  hours  nor 
by  desiccation  extending  over  several  months. 

Virulence. — The  virulence  of  the  organism  is  preserved  by  the  spore  :  this 
virulence  is  maintained  indefinitely  in  cultures,  but  for  purposes  of  inocula- 
tion it  is  always  necessary  to  make  a  new  sub-culture,  since  the  spores  alone 
in  ordinary  doses  are  harmless  and  the  toxin  is  weakened  by  keeping  (infra). 
If  this  precaution  be  not  observed  the  organism  may  be  erroneously  thought 
to  have  become  attenuated. 

All  strains  of  the  bacillus  are  not  equally  virulent :  some  are  non- virulent 
even  for  guinea-pigs  (vide  ante],  but  as  has  already  been  pointed  out  these 
variations  are  not  stable,  for  the  virulence  of  a  slightly  virulent  bacillus 
may  be  easily  raised  by  passage  through  guinea-pigs. 

Leclainche  and  Vallee  have  shown  that  the  method  of  attenuation  by  heat  devised 
by  Arloing,  Cornevin  and  Thomas  for  the  bacillus  of  quarter  ill  (Chap.  XXXVII. ),  is 
equally  applicable  to  this  bacillus.  Heat  the  blood  of  an  animal  dead  of  malignant 
oedema  in  sealed  ampoules  at  37°  C.  for  5  days  and  treat  the  contents  of  the  ampoules 
by  Arloing's  method  (p.  556).  The  resulting  powder  constitutes  an  attenuated  virus 
with  which  animals  may  be  immunized  against  the  disease. 

2.  Toxin. 

(i)  As  early  as  1887  Eoux  and  Chamberland  studied  the  toxin  which  the 
bacillus  produces  in  cultures  and  in  the  living  organism. 


566  THE   BACILLUS   OF  MALIGNANT  (EDEMA 

After  inoculation  the  vibrio  multiplies  and  rapidly  invades  all  the  tissues  of  the 
body  :  it  is  not  therefore  to  be  expected  that  its  toxin  will  be  as  powerful  as  that  of 
micro-organisms  which  like  the  tetanus  bacillus  and  the  diphtheria  bacillus  only 
grow  at  the  site  of  inoculation.  While  the  toxins  of  these  latter  organisms  are 
fatal  to  small  laboratory  animals  in  almost  infinitesimal  doses,  filtered  cultures  of 
the  bacillus  of  malignant  oedema  have  to  be  inoculated  in  doses  of  several  c.c.  to 
produce  a  similar  result. 

Roux  and  Chamberland  by  filtering  through  a  porcelain  bougie  an  emulsion 
made  with  the  muscles  of  guinea-pigs  and  rabbits  which  had  died  of  the 
disease  obtained  a  liquid  which  proved  fatal  when  inoculated  in  quantities 
of  40  c.c.  into  the  peritoneal  cavity  of  a  guinea-pig. 

(ii)  Besson  re-investigated  the  toxin  of  the  bacillus.  Cultures  and  the 
exudate  from  animals  which  had  just  died  of  the  acute  experimental  disease 
were  used  after  being  filtered  through  a  Chamberland  bougie. 

(a)  For  cultures,  it  was  necessary  to  select  a  medium  favouring  the  forma- 
tion of  the  largest  quantity  possible  of  toxin.  Cultures  in  ordinary  broth 
were  not  suitable.  Better  results  were  obtained  with  cultures  grown  on 
meat  pulp. 

Besson's  method  for  the  preparation  of  the  toxin. — To  500  grams  of  minced  beef 
in  a  flask  of  1500  c.c.  capacity  and  plugged  with  wool  add  a  few  c.c.  of  a  1  per  cent, 
solution  of  soda  and  heat  at  115°  C.  in  the  autoclave  for  20  minutes.  When  cool,  sow 
the  medium  with  a  little  of  the  exudate  obtained  from  a  guinea-pig  which  has  died 
as  the  result  of  the  inoculation  of  a  bacillus  the  virulence  of  which  has  been  increased 
by  passage  through  guinea-pigs.  For  the  cotton-wool  plug  substitute  a  sterile 
india-rubber  cork  carrying  two  tubes,  one  of  which  dips  into  the  contents  of  the  flask 
and  outside  is  bent  at  an  acute  angle  and  terminates  in  a  fine  point :  this  tube  serves 
for  the  decantation  of  the  toxin  after  incubation.  The  other  tube  only  passes  just 
through  the  cork  and  outside  is  bent  at  a  right  angle,  plugged  with  wool,  and  con- 
stricted near  the  end.  Attach  the  latter  tube  to  the  water  pump  and  when  a  vacuum 
is  created  seal  the  tube  in  the  blow-pipe  at  the  constricted  part.  Incubate  the 
flask  at  37°  C.  After  incubating  about  20  hours  numerous  bubbles  of  gas  burst 
on  the  surface  of  the  doughy  mixture  in  the  flask,  the  meat  has  a  characteristic  bright 
pink  colour  and  tends  to  divide  into  two  layers,  i.e.  the  culture  consists  of  a  semi- 
solid  irregular  broken  mass  bathed  in  a  reddish  turbid  fluid.  A  considerable  amount 
of  gas  is  formed  during  incubation  and  if  allowed  to  accumulate  will  check  the  growth 
of  the  organism  and  prevent  the  formation  of  toxin  ;.  it  is  therefore  advisable  after 
about  48  hours  to  break  off  the  sealed  end  of  the  tube,  when  the  gas,  which  has  a 
most  offensive  odour,  will  escape  under  considerable  pressure.  The  tube  need  not 
be  sealed  again  because  the  amount  of  gas  formed  is  sufficient  to  keep  the  culture 
under  anaerobic  conditions. 

Experience  has  shown  that  the  toxin  content  is  greatest  about  the  sixth  day  of 
incubation  and  afterwards  rapidly  diminishes,  so  that  the  flask  must  then  be  taken 
out  of  the  incubator.  Decant  the  liquid  and  press  the  solid  portion  in  the  meat 
press.  Add  the  juice  obtained  from  the  latter  to  the  liquid  and  filter  the  mixture 
through  a  Chamberland  bougie. 

This  toxin  is  more  active  than  E-oux  and  Chamberland's.  A  dose  of 
3-5  c.c.  injected  into  the  peritoneal  cavities  of  guinea-pigs  weighing  450-600 
grams  produces  symptoms  similar  to  those  seen  in  the  last  stages  of  the 
septicsemic  form  of  the  disease  but  from  which  the  animals  rapidly  recover. 
Similar  or  larger  doses  inoculated  into  the  sub-cutaneous  tissues  have  much 
less  effect  on  the  general  condition  and  hardly  any  effect  at  all  on  the  tem- 
perature, but  give  rise  to  a  local  oedema  or  slough. 

Intra-peritoneal  inoculation  of  doses  of  5-10  c.c.  is  rapidly  fatal  to  guinea- 
pigs  weighing  300-400  grams  and  the  symptoms  are  not  preceded  by  any 
incubation  period. 

Guinea-pigs  and  rabbits  inoculated  frequently  with  small  doses  of  toxin 
suffer  as  a  rule  from  a  chronic  intoxication  and  ultimately  die. 


VACCINATION  567 

Post  mortem  examination  of  animals  dying  as  the  result  of  the  intra-peri- 
toneal  inoculation  of  toxin  shows  the  intestines  and  peritoneum  to  be  congested 
and  the  peritoneal  cavity  to  contain  a  little  sterile  exudate. 

The  addition  of  iodine  solution  seems  to  modify  the  properties  of  the  toxin  very 
slightly.  Heat,  on  the  other  hand,  has  a  distinct  effect :  a  temperature  of  80° 
or  100°  C.  markedly  diminishes  the  toxic  property  of  cultures. 

If  the  toxin  be  kept  for  some  time  at  35°  C.  in  diffused  daylight  it  soon  loses  its 
properties,  but  if  stored  in  a  closed  vessel  away  from  air  and  light  at  room  temperature 
it  does  not  deteriorate. 

(6)  The  product  obtained  by  filtering  the  oedematous  fluid  of  animals  dead 
of  the  disease  is  much  less  toxic  than  the  toxin  prepared  as  above.  Doses 
of  2-10  c.c.  of  the  filtered  exudate  inoculated  into  the  peritoneal  cavities  of 
guinea-pigs  weighing  280-350  grams  do  the  animals  no  harm.  In  guinea- 
pigs  weighing  300  grams  doses  of  15-20  c.c.  produce  more  or  less  severe 
symptoms  but  the  animals  always  recover.  Death  only  occurs  when  doses 
of  30-40  c.c.  are  given  intra-peritoneally. 

Chemio tactic  properties. — The  toxin  of  the  bacillus  of  malignant  oedema 
has  negative  chemiotactic  properties  (Besson). 

Aspirate  the  toxin  into  capillary  tubes  2-3  cm.  long  and  seal  one  end.  Introduce 
the  tubes  beneath  the  skin  of  guinea-pigs  and  rabbits  through  very  small  incisions 
and  after  8,  10  and  20  hours  remove  them  and  examine  their  contents.  Although 
control  tubes  containing  a  little  of  the  broth  used  for  the  cultures  and  inserted 
beneath  the  skin  at  the  same  time  now  contain  a  turbid  liquid  very  rich  in  leucocytes 
the  contents  of  the  tubes  containing  the  toxin  are  clear  and  no  leucocytes  can  be 
detected  on  microscopical  examination.  It  is  only  after  24  or  30  hours  that  the 
latter  contain  leucocytes  :  this  may  be  due  either  to  the  fact  that  the  properties 
of  the  toxin  have  undergone  modification  from  their  prolonged  contact  with  the 
living  tissues  or  to  the  fact  that  the  toxin  has  diffused  out  and  been  replaced  by 
lymph. 

Heating  at  85°  C.  for  2  or  3  hours  fundamentally  alters  the  chemiotactic 
properties  of  the  toxin  :  previously  negative  they  are  now  positive  and 
tubes  inserted  beneath  the  skin  of  guinea-pigs  and  rabbits  quickly  fill  with 
leucocytes. 

(iii)  Leclainche  and  Morel  obtained  an  active  toxin  by  growing  the  organism 
in  Martin's  broth  :  the  culture  was  decanted  not  filtered,  because  the  filter 
retained  a  portion  of  the  toxin.  The  product  obtained  killed  rabbits  in 
doses  of  5  c.c.  when  inoculated  intra-venously.  Intra-cerebrally  5-6  drops 
produced  a  fatal  result. 

3.  Vaccination. 

Roux  and  Chamberland  succeeded  in  vaccinating  guinea-pigs  by  repeatedly 
inoculating  large  doses  of  broth  cultures  heated  at  110°  C.  for  10  minutes 
into  the  peritoneal  cavity.  After  inoculating  a  total  quantity  of  120  c.c. 
of  the  heated  culture  on  three  separate  occasions  at  intervals  of  3  days  the 
animals  were  found  to  be  immune. 

Immunization  by  the  injection  of  increasing  doses  of  filtered  meat  cultures 
is  very  difficult  (Besson).  Most  of  the  animals  submitted  to  the  treatment 
died  of  a  chronic  cachexia. 

Roux  and  Chamberland  immunized  guinea-pigs  by  inoculating  them  on 
seven  or  eight  occasions  with  1  c.c.  of  oedema  fluid  which  had  been  filtered 
through  a  porcelain  bougie. 

Besson  has  succeeded  in  immunizing  rabbits  by  repeatedly  inoculating 
them  in  the  cellular  tissue  of  the  ear  with  the  unaltered  exudate.  On  the 
first  occasion  ^-lih  of  a  drop  is  inoculated  into  the  extreme  tip  of  the  ear : 
a  sharp  reaction  occurs  and  the  inoculated  part  has  an  erysipelatous  appear- 


568  THE   BACILLUS   OF   MALIGNANT  (EDEMA 

ance.  The  inoculations  are  repeated  every  week  or  10  days,  gradually 
increasing  the  doses  inoculated  to  j,  i  and  1  drop,  and  inoculating  each  time 
a  little  nearer  the  base  of  the  ear.  After  5  or  7  weeks  the  animal  may  be 
inoculated  with  1  drop  of  virulent  exudate  beneath  the  skin  of  the  abdominal 
wall,  and  the  degree  of  immunity  is  increased  by  successive  inoculations  into 
the  cellular  tissue  of  the  trunk.  When  large  doses  are  being  administered 
it  is  not  uncommon  to  find  that  an  abscess  forms  in  which  phagocytosis  is 
very  active  :  these  abscesses  resolve.  The  immunity  so  acquired  is  per- 
manent and  may  be  transmitted  from  the  mother  to  the  offspring. 

It  should  be  mentioned  that  Leclainche  and  Vallee  have  succeeded  in 
immunizing  guinea-pigs  by  Aiioing's  vaccination  method  (supra). 

4.  Serum  therapy. 

Leclainche  obtained  a  very  powerful  serum  by  inoculating  asses,  animals 
which  are  only  slightly  susceptible  to  the  disease,  intra-venously  and  then 
intra-muscularly  (into  several  muscles)  first  with  the  exudate  and  afterwards 
with  cultures  grown  in  Martin's  broth. 

This  serum  is  powerfully  antitoxic  and  neutralizes  the  toxin  of  the  bacillus. 

A  mixture  of  2  c.c.  of  the  serum  with  5  drops  of  an  exudate  is  harmless  to 
guinea-pigs.  The  animals  show  no  degree  of  immunity  as  a  result,  and  die 
as  quickly  as  the  controls  when  tested  by  inoculation.  The  serum  is  without 
action  on  the  virus  of  quarter  ill. 

5.  Agglutination. 

Leclainche's  serum  agglutinates  young  cultures  grown  in  Martin's  broth 
in  a  few  minutes.  This  agglutination  occurs  in  dilutions  of  1  in  30  to  1  in 
3,000  and  is  more  marked  under  aerobic  than  under  anaerobic  conditions. 
The  bacillus  of  quarter  ill  is  not  agglutinated  by  a  malignant  oedema 
serum. 


CHAPTER  XXXIX. 

CERTAIN  ANAEROBIC  MICRO-ORGANISMS  FOUND 
IN  GANGRENOUS  SUPPURATIONS. 

I. — Bacillus  perfringens,  p.  569. 

Ghon  and  Sachs'  bacillus,  p.  571. 
II. — Bacillus  pseudo-oedema,  p.  571. 
III. — Bacillus  ramosus,  p.  571. 
IV. — Bacillus  serpens,  p.  572. 
V. — Bacillus  thetoides,  p.  572. 
VI.— Bacillus  fragilis,  p.  573. 
VII. — Bacillus  fusiformis,  p.  574. 
VIII. — Spirillum  nigrum,  p.  577. 
IX. — Staphylococcus  parvulus,  p.  578. 
X. — Micrococcus  fcetidus,  p.  578. 
XI. — Bacillus  aerobicus  sepis,  p.  578. 

THE  study  of  the  pathogenic  anaerobic  micro-organisms  has  until  recently 
been  much  neglected,  but  thanks  to  the  work  of  Veillon  and  his  pupils,  interest 
in  this  branch  of  bacteriology  has  now  been  aroused.  A  rich  bacterial  flora 
has  been  found  in  gangrene  and  in  gangrenous  suppurations  and  it  is  desirable 
that  the  principal  species  isolated  should  be  shortly  described  here. 


I.   BACILLUS   PERFRINGENS. 

THE  Bacillus  perfringens  is  an  anaerobic  organism  discovered  in  1898  by 
Veillon  and  Zuber  in  conditions  of  gangrene.  Certain  organisms  which  were 
previously  known  and  to  which  various  names  were  given  are  now  believed 
to  be  identical  with  the  Bacillus  perfringens  :  the  following  must  be  regarded 
as  coming  within  this  category  : — 

1.  The    bacillus    discovered    by    Achalme    in    1891    in    acute    articular 
rheumatism  ; 

2.  The  Bacillus  aerogenes  capsulatus  of  Welch,  which  was  found  in  the 
tissues  of  a  dead  body  ; 

3.  The  Bacillus  phlegmonis  emphysematosce  isolated  by  Frsenkel  from  a 
phlegmonous  inflammation. 

The  Bacillus  perfringens  is  exceedingly  wide- spread.  Achalme  found  it  in  the 
blood  of  persons  suffering  from  rheumatism  and  in  the  myocardium  of  two  individuals 
who  had  died  of  acute  articular  rheumatism  ;  Veillon  and  Zuber  in  gangrenous 
suppurations  (appendicitis,  etc.)  ;  Guyon,  Albarran,  Jungano,  in  urinary  abscesses  ; 
Frsenkel  in  an  inflammatory  swelling  ;  Guillemot  in  a  case  of  gaseous  gangrene  ; 
Jungano  in  cases  of  chronic  urethritis  ;  Chaillous  and  Benedetti  in  ocular  infections. 


570  ANAEROBIC  ORGANISMS  IN  GANGRENE 

It  is  a  normal  inhabitant  of  the  alimentary  canal  of  man  and  many  of  the  lower 
animals  and  it  possibly  plays  a  part  in  the  aetiology  of  certain  forms  of  diarrhoea 
(Tissier,  Metchnikoif).  It  is  also  present  in  bodies  undergoing  decomposition. 

1.  Experimental  inoculation. 

Guinea-pigs  are  the  animals  most  susceptible  to  experimental  inoculation. 
Death  follows  sub-cutaneous  inoculation  in  24-48  hours  with  lesions  similar 
to  those  of  malignant  oedema  :  at  the  site  of  inoculation  the  skin  is  stripped 
up  by  a  gas-containing  abscess :  the  internal  organs  are  crowded  with  micro- 
organisms. 

Rabbits  are  not  so  susceptible.  Sub-cutaneous  inoculation  is  followed  by 
the  formation  of  a  large  gas-containing  abscess  which  generally  resolves  : 
if  death  occur  it  does  not  usually  take  place  until  about  a  week  after  the 
inoculation.  Inoculation  into  the  veins  even  is  not  always  fatal. 

2.  Morphology. 

The  Bacillus  perfringens  is  a  large,  straight,  non-motile  bacillus  of  variable 
length  and  a  little  larger  than  the  anthrax  bacillus.  The  ends  are  square 

cut  or  slightly  rounded.  In  the  tissues  the 
bacilli  are  as  a  rule  shorter  than  in  cultures,  are 
often  surrounded  by  a  very  distinct  capsule,  and 
are  occasionally  (e.g.  in  the  peritoneal  exudate) 
arranged  in  somewhat  long  chains.  In  cultures 
on  liquid  media  the  bacilli  are  generally  long 
and  slender  :  in  old  cultures  involution  forms 
occur — deformed  bacilli  with  rounded  ends  and 
staining  irregularly. 

Spore  formation  does  not  occur  in  sugar-con- 
taining  media  but  in  media  containing  no  sugar 
and  especially  on  cooked  white  of  egg  in  normal 
FIG.   267.— Bacillus  perfringens.   saline  solution  an  oval  spore  is  formed  towards 
CarLiSn1nglTfooo°th  ^^   one   end  whicn  stains  with  difficulty  (Musca- 

tello). 

Staining  reactions. — The  Bacillus  perfringens  stains  easily  with  the  basic 
aniline  dyes  and  is  gram-positive. 

3.  Cultural  characteristics. 

The  Bacillus  perfringens  is  a  strictly  anaerobic  organism  and  grows  best 
on  media  containing  glucose.  At  37°  C.  and  even  at  ordinary  temperatures 
it  grows  very  rapidly.  It  decomposes  powerfully  sugars  and  proteins  giving 
off  a  considerable  quantity  of  gas  which  has  an  odour  of  butyric  acid.  It 
produces  no  indol.  According  to  Achalme  it  reduces  nitrates  to  nitrites. 

The  vitality  of  the  bacillus  is  rather  low  and  it  should  be  frequently  sub- 
cultivated. 

Rosenthal  claims  to  have  been  able  to  adapt  the  Bacillus  perfringens  to  aerobic 
conditions.  Rosenthal  distinguishes  two  varieties  of  the  bacillus,  the  common 
variety  which  grows  with  a  fetid  odour,  and  a  variety  which  does  not  give  rise  to 
this  disagreeable  smell  and  is  less  active  in  attacking  culture  media :  the  latter 
variety  is  said  to  correspond  to  Achalme' s  rheumatism  bacillus. 

Broth. — The  medium  soon  becomes  cloudy  and  later  the  growth  precipitates 
in  the  form  of  whitish  flakes  leaving  the  broth  clear. 

Gelatin. — In  gelatin  containing  no  sugar,  some  strains  distinctly  liquefy 
the  medium  while  with  others  liquefaction  only  takes  place  slowly  and  to  a 
slight  degree. 


BACILLUS   PSEUDO-CEDEMA  571 

Discrete  colonies  are  round  with  irregular  margins,  and  are  slightly  granular  : 
the  medium  is  split  by  bubbles  of  gas  and  liquefaction  then  occurs. 

Agar. — According  to  Jungano  and  Distaso,  isolated  colonies  on  agar  are 
characteristic  :  they  are  small,  lenticular  or  heart-shaped,  with  regular  sharp- 
cut  edges,  and  under  the  microscope  are  somewhat  granular.  Numerous 
bubbles  of  gas  are  rapidly  formed. 

Milk. — Coagulation  takes  place  in  24  hours  and  is  accompanied  by  a  smell 
of  butyric  acid. 

White  of  egg. — White  of  egg  is  attacked  very  slowly  and  a  black  pigment 
is  formed  at  the  bottom  of  the  tube. 

4.  Toxin. 

Cultures  sterilized  by  filtration  or  by  chloroform  have  no  action  on  guinea- 
pigs  or  rabbits  (Jungano). 

Korentchevsky  obtained  a  toxin  from  a  bacillus  isolated  from  a  dog  the 
virulence  of  which  had  been  increased  by  passage  through  three  rabbits.  This 
toxin  was  fatal  to  rabbits  in  quantities  of  1  c.c.  per  kilogram  of  body  weight. 

GHON  AND  SACHS5  BACILLUS. 

Ghon  and  Sachs  found  a  bacillus  resembling  the  Bacillus  perfringens  in  the  liver 
of  a  person  affected  with  gaseous  gangrene.  This  bacillus,  however,  was  more 
slender  than  the  B.  perfringens  and  was  sometimes  motile  and  curved :  the  spore 
was  situated  in  the  middle  and  took  Gram's  stain  when  young.  The  organism  is 
very  slightly  pathogenic  for  laboratory  animals  (mice,  guinea-pigs  and  rabbits), 
in  which  it  produces  a  temporary  swelling. 

II.  BACILLUS  PSEUDO-CEDEMA. 

The  Bacillus  pseudo-oedema  was  first  isolated  by  Liborius  from  garden 
soil  and  afterwards  by  Sanfelice  from  soil  and  the  excreta  of  animals.  The 
Proteus  hominis  capsulatus  obtained  by  Bordoni  Uffreduzzi  from  a  case  of 
human  septicaemia  is  apparently  the  same  organism. 

Experimental  inoculation. — The  B.  pseudo-osdema  is  pathogenic  for  rabbits, 
guinea-pigs  and  mice  ;  if  a  considerable  quantity  of  culture  be  inoculated 
the  animals  suffer  from  lesions  similar  to  those  of  malignant  oedema. 

Morphology. — The  bacillus  is  stouter  than  the  bacillus  of  malignant  oedema 
and  sometimes  forms  filaments.  It  has  a  very  distinct  capsule,  and  generally 
shows  two  oval  terminal  spores.  It  stains  easily  with  the  aniline  dyes  and 
irregularly  with  Gram's  stain. 

Cultural  characteristics. — The  Bacillus  pseudo-oedema  is  a  strict  anaerobe 
and  grows  abundantly  on  the  ordinary  culture  media  producing  a  considerable 
quantity  of  gas  and  giving  off  a  fetid  odour.  It.  liquefies  gelatin. 

III.  BACILLUS  RAMOSUS. 

This  bacillus  was  found  by  Veillon  and  Zuber  in  a  number  of  instances  in 
pus  from  gangrenous  inflammations  (otitis,  appendicitis,  etc.),  and  by  Monnier 
in  dental  caries.  It  is  a  normal  inhabitant  of  the  intestine.  The  bacillus 
described  by  Lotti  in  a  case  of  appendicitis,  Grigoroff's  "  A  "  bacillus  (appendi- 
citis), and  the  Bacillus  posciloides  of  Roger  and  Gamier  are,  according  to 
Jungano  and  Distaso,  the  same  organism. 

Experimental  inoculation. — The  Bacillus  ramosus  is  pathogenic  for  rabbits, 
guinea-pigs  and  mice  ;  these  animals  die  in  6-8  days  after  being  inoculated 
sub-cutaneously  with  cultures. 


572  ANAEROBIC  ORGANISMS  IN  GANGRENE 

Morphology. — The  Bacillus  ramosus  is  a  small  slender  non-motile  bacillus, 
a  little  larger  than  the  bacillus  of  mouse  septicaemia,  occurring  singly,  or  in 
pairs  parallel  to  one  another  or  at  an  acute  angle.  In  cultures  it  is  often 

longer,  and  then  has  somewhat  the  appearance 
of  the  diphtheria  bacillus.  Occasionally  the 
bacilli  are  arranged  end  to  end  forming  long 
filamentous  chains  ;  and  branching  forms  have 
been  described.  It  does  not  appear  to  produce 
spores. 

L  "\  '          *^^  Staining    reactions.  - —  The    Bacillus    ramosus 

(       ^^-v     1\    C         —      stains    easily   with    carbol-violet  and    is    gram- 
J.  '  S-  positive. 

Cultural  characteristics. — The  Bacillus  ramosus 
is  .a  strict  anaerobe ;    it  grows  on  the  ordinary 
media  and  best  between  33°  C.  and  39°  C.     Cul- 
tures grow  slowly  and  are  scanty  and  have  a  fetid 
ZuberTSM         odour.     The  bacillus  produces"  a  little  gas  and 

retains  its  vitality  for  a  long  time. 
Broth. — Uniform  turbidity. 
Gelatin. — No  growth. 

Agar. — Isolated  colonies  are  small,  rounded  or  cuneiform,  with  regular 
edges. 

Milk. — Milk  is  coagulated  ;    the  casein  is  not  attacked. 


IV.   BACILLUS  SERPENS. 

The  Bacillus  serpens  was  found  by  Veillon  and  Zuber  in  pus  from  a  mastoid 
abscess,  by  Hist  and  Guillemot  in  gangrene  of  the  lung,  and  by  other  observers 
in  similar  morbid  processes. 

Experimental  inoculation. — The  Bacillus  serpens  is  pathogenic  for  rabbits, 
guinea-pigs  and  mice.  In  guinea-pigs,  sub-cutaneous  inoculation  is  followed 
by  the  formation  of  a  fetid  abscess  and  death  takes  place  in  about  a  week. 

Morphology. — The  Bacillus  serpens  occurs  as  large  straight  rods  with 
rounded  ends.  In  cultures  the  bacilli  are  often  arranged  in  pairs  end  to  end, 
are  motile  and  move  with  a  sort  of  an  undulatory  movement. 

Staining  reactions. — The  Bacillus  serpens  stains  easily  with  the  basic  aniline 
dyes  containing  a  mordant.  It  is  gram-negative. 

Cultural  characteristics. — The  Bacillus  serpens  grows  anaerobically  on  all 
the  ordinary  media  and  at  the  temperature  of  the  laboratory.  Cultures  have 
a  fetid  odour  and  give  off  a  little  gas. 

Broth. — Broth  soon  becomes  cloudy  but  the  growth  subsequently  precipi- 
tates and  the  medium  slowly  clears. 

Gelatin. — Small  round  greyish  colonies  appear  about  the  fourth  or  fifth 
day.  The  medium  is  slowly  liquefied. 

Agar. — After  about  24  hours,  isolated  colonies  appear  as  small,  grey,  round, 
granular  points,  translucent  at  first  but  opaque  later. 


V.   BACILLUS  THETOIDES   vel   FUNDULIFORMIS. 

This  organism  was  found  by  Halle  in  the  vagina  and  in  the  pus  in  cases  of 
inflammation  of  Bartholin's  glands,  and  by  Rist  and  Guillemot  in  pulmonary 
gangrene,  mastoid  abscesses,  etc. 

The  Bacillus  thetoides  is  described  by  Veillon  and  Zuber  as  B.  funduliformis. 


BACILLUS   FRAGILIS  573 

Experimental  inoculation.  —  Guinea-pigs  appear  to  be  the  only  laboratory 
animals  susceptible  to  infection  with  the  bacillus. 

Morphology.  —  -The  Bacillus  thetoides  is  a  pleomorphic  organism.     In  the 
tissues,  it  generally  occurs  as  a  slender  fairly  straight  rod  :   in  cultures,  rods, 
filaments,  and   forms  with  terminal  enlarge- 
ments are  found.     It  is  non-motile. 

Staining  reactions.  —  The   Bacillus  thetoides  Q       f==^ 

stains  badly  with  the  basic  aniline  dyes  :  por- 
tions  of  the  organism  remain  unstained  and  & 

particularly  the  ends  so  that  the  bacillus  not 


uncommonly  resembles  the  Greek  letter  0.    It  g       _ 

is  gram-negative.  <^     I  <,       fl 

Cultural  characteristics.—  The  Bacillus  the-  ft  ^ 

toides  is   a   strictly   anaerobic  organism  and  \ 

does  not  grow  at  temperatures  below  22°  C.—  /     \ 

25°  C.     It  produces  no  gas. 

It  grows  on  agar  at  22°  C.  but  not  on  gelatin.    &'S^S^^!!g 
Agar  appears  to  be  the  best  medium  :  on  this 
medium  the  growth  of  the  organism  takes  the  form  of  small  rounded  homo- 
geneous pale  yellow  almost  punctiform  colonies  with  regular  margins. 


VI.   BACILLUS  FRAGILIS. 

This  bacillus  was  isolated  by  Zuber  and  Veillon  from  some  pus  from  a  case 
of  appendicitis  and  it  has  since  been  frequently  found  in  pus  from  gangrenous 
conditions  of  the  appendix  (Veillon  and  Zuber,  Grigorofi),  in  peri-urethral 
infections  (Cottet,  Jungano),  in  pulmonary  gangrene  (Guillemot),  in  dental 
caries  (Monnier),  etc. 

Experimental  inoculation. — The  Bacillus  fragilis  is  not  very  pathogenic  for 
laboratory  animals. 

A  gangrenous  inflammation  generally  follows  the  sub-cutaneous  inoculation 
of  the  bacillus  into  a  guinea-pig  and  the  animal  may  die  from  20-30  days 

later.     The  inoculation  of  large  doses  of  cultures 
*^!        •«*  into  the  veins    of  rabbits   leads  to  death   from 

**  cachexia  ;    there  is  no  multiplication  of   the  or- 

**        ganism. 

\    •-*  Morphology  .—The   Bacillus  fragilis  is  a  small 

-  ^  short   non-motile   organism   with   rounded    ends 

«•*        I       ""  jf  sometimes  having  the  appearance  of  a  diplococcus : 

*"*•  ^.  it  is  generally  longer  in  culture  than  in  the  tissues. 

\  •     /          ^      It  does  not  form  spores. 

9     f  '     -          Staining  reactions. — The  Bacillus  fragilis  stains 

with  some  difficulty  with  the  basic  aniline  dyes 
containing  a  mordant,  so  that  in  stained  films  it 

FIG.  270.— Bacillus  fragilis.  Film  frequently  has  a  granular  appearance  due  to  parts 
ffu°chsina  bx%hooculture'    Carbo1'  of  the  organism  not  having  taken  the  stain.     It 

is  gram-negative. 

Cultural  characteristics. — The  Bacillus  fragilis  is  a  strict  anaerobe  and 
grows  slowly  and  feebly  with  a  disagreeable  smell  and  the  production  of  a 
small  quantity  of  gas.  Cultures  have  very  little  vitality  and  die  if  kept  in 
the  incubator  for  6-8  days. 

Broth. — The  medium  becomes  cloudy  about  the  third  day. 

Gelatin. — Small  punctiform  colonies  appear  in  about  10  days  to  a  fortnight. 


574  ANAEROBIC   ORGANISMS  IN  GANGRENE 

Agar. — Very  small  translucent  colonies  often  having  a  muriform  appearance 
are  visible  about  the  third  or  fourth  day  (Jungano  and  Distaso). 


VII.   BACILLUS  FUSIFORMIS. 
1.  Introduction. 

The  Bacillus  fusiformis  was  discovered  by  Vincent  in  cases  of  Hospital 
gangrene. 

The  bacillus  is  constantly  found  in  the  lesions  of  hospital  gangrene  being  present 
in  very  large  numbers  in  the  pseudo-membranous  tissue  covering  the  surface  of 
the  wounds.  It  does  not  invade  the  tissues  and  is  never  found  in  the  blood  or 
lymphatic  glands. 

In  the  lesions  the  Bacillus  fusiformis  may  be  found  in  pure  culture  or  associated 
with  other  organisms  :  sometimes  micrococci  or  bacilli  are  found  (especially  on 
the  surface  of  the  lesions)  but  the  organism  most  commonly  associated  with  the 
bacillus  is  a  spirillum  (40  times  out  of  47  cases  examined)  which  is  very  delicate 
and  difficult  to  stain  (vide  infra).  Other  organisms  which  may  be  found  are  :  staphy- 
lococci,  streptococci,  proteus  vulgaris,  bacillus  pyocyaneus,  bacillus  coli,  pneumobacillus. 

The  bacillus  has  also  been  found  by  the  same  observer  in  cases  of  sore 
throat  (Vincent's  angina),  in  diphtheroid  stomatitis  (accompanied  by  various 
organisms)  and  in  membranous  stomatitis  associated  with  a  spirillum.1 
Vincent's  observations  have  been  confirmed  by  Bertheim,  Raoult  and  Thiry, 
Abel  and  others. 

The  bacilli  described  by  Veillon  and  Zuber  and  by  Grigoroff  and  Perrone 
in  appendicitis,  by  Bernheim  and  Popischill  in  gangrenous  laryngitis,  by 
Silberschmidt  in  fetid  bronchitis,  by  Freimuth  and  Petruschy,  Passini,  Leiner 
and  others  in  noma,  as  well  as  the  organism  described  by  Zeitz  as  the 
Bacillus  hastilis  and  found  by  him  in  the  crypts  of  the  tonsil,  are  all  identical 
with  the  Bacillus  fusiformis. 

The  Bacillus  fusiformis  has  been  shown  to  be  present  in  the  mouths  of 
healthy  persons  and  in  the  tartar  on  the  teeth  by  Muhlens  and  others. 

2.  Experimental  inoculation. 

A.  Hospital  gangrene.  Man. — Direct  inoculation  from  man  to  man 
attempted  long  ago  by  Willaume  and  others,  and  more  recently  by  Vincent 
on  himself  and  on  a  number  of  Arabs  has  always  failed  to  set  up  the  lesions 
of  hospital  gangrene. 

Animals. — 1.  In  guinea-pigs,  rabbits  and  white  rats  artificial  wounds  covered 
with  fresh  pieces  of  membrane  have  healed  rapidly  without  any  of  the  features 
of  hospital  gangrene.  The  inoculation  of  emulsions  of  false  membranes 
either  beneath  the  skin  or  into  the  peritoneum,  blood,  or  muscles  leads  to 
nothing  more  serious  than  an  abscess  due  to  the  other  organisms  present. 
Inoculation  fails  even  after  cutting  the  sciatic  nerve,  tying  the  femoral  artery 
or  crushing  the  limb. 

Coyon,  however,  succeeded  in  producing  an  infection  in  a  guinea-pig.  He 
lacerated  the  muscles  of  the  thigh  of  the  animal  and  made  a  deep  ragged 
opening  into  which  he  introduced  the  pseudo-membrane  from  a  case  of 
hospital  gangrene.  The  wound  was  sutured  and  the  skin  painted  over  with 
collodion.  A  funnel-shaped  wound  developed  covered  with  a  tough  mem- 
brane in  which  the  Bacillus  fusiformis  was  present  in  enormous  numbers. 

Healthy  animals  even  when  fasting  are  not  susceptible. 

1  This  spirillum  will  be  referred  to  later  in  connexion  with  the  spirochsete  of  syphilis 
which  it  resembles  closely. 


BACILLUS   FUSIFORMIS 


575 


2.  By  operating  upon  animals  whose  resistance  had  been  lowered  by  a 
micro-organic  disease,  or  by  mixing  the  bacillus  in  the  emulsion  with  other 
organisms  Vincent  succeeded  in  producing  hospital  gangrene. 

A  tuberculous  rabbit  was  inoculated  sub-cutaneously  in  the  flank  with  1  c.c.  of  an 
emulsion  of  gangrenous  material :  at  first  a  small  abscess  formed,  and  later  an  ulcer 
appeared  covered  with  a  membrane  containing  the  bacillus. 

Hospital  gangrene  has  been  produced  in  rabbits  by  mixing  the  virus  with  a  few 
drops  of  a  culture  of  streptococci,  staphylococci,  bacillus  coli,  bacillus  of  Friedlander 
or  bacillus  pyocyaneus.  In  these  lesions  the  ancillary  organism  always  tends  to  dis- 
appear leaving  the  specific  organism  in  practically  pure  culture.  Most  frequently 
the  ancillary  organisms  occur  on  the  surface  of  the  lesions  while  the  Bacillus  fusi- 
formis  predominates  in  the  deeper  layers  of  the  exudate.  Inoculations  from  animal 
to  animal  do  not  succeed. 

B.  Vincent's  angina. — Inoculation  of  the  false  membranes  from  cases  of 
Vincent's  angina  beneath  the  skin  or  into  the  muscles  of  laboratory  animals 
produces  abscesses  and  ulcerating  foci  of  necrosis  in  which  the  Bacillus  fusi- 
formis together  with  many  other  organisms  is  found.     The  inoculation  at  the 
same  time  of  a  1  in  5  solution  of  lactic  acid  stimulates  the  formation  of  the 
lesions  and  the  growth  of  the  bacillus. 

The  inoculation  of  impure  cultures  obtained  by  sowing  pieces  of  the  false 
membranes  in  Martin's  broth  gives  rise  to  similar  lesions. 

C.  Pure  cultures. — The  inoculation  of  pure  cultures  of  the  bacillus  from 
whatever   source     derived  is  generally  followed  by  negative  results ;    the 
pathogenic  power  of  different  strains  varies  and  in  any  case  rapidly  disappears 
in  sub-cultures  (Ellermann). 

Muhlens,  and  Tunnicliffe  have  invariably  had  negative  results  :  the  former 
inoculated  pure  cultures  (5th  and  7th  generations)  intra-venously,  intra- 
peritoneally,  and  sub-cutaneously  into  rabbits,  guinea-pigs,  and  mice  and 
only  once  obtained  a  small  abscess  in  a  rabbit. 

Leiner  and  Kepaci  investigated  some  strains  which  were  pathogenic  for 
rabbits  and  mice.  Ellermann  produced  suppuration  but  not  necrosis. 

3.  Microscopical  appearance  and  staining  reactions. 

Microscopical  appearance. — The  Bacillus  fusiformis  is  a  long  rod-shaped 
organism  measuring  5-10/^  x  0'6-0'8/x  slightly  swollen  in  the  middle  and 
pointed  at  the  ends  ;  it  is  non-motile. 

In  films  prepared  from  the  false  membranes 
Vincent's  angina  and  stained  with  carboi-fuchsin 
or  one  of  the  carbol-violet  stains,  numerous 
bacilli  of  the  type  described  above  will  be  seen, 
often  straight  but  sometimes  slightly  curved  or 
assuming  the  form  of  an  elongated  S.  Many  of 
the  bacilli  are  arranged  in  pairs.  The  appear- 
ance of  these  organisms  recalls  to  some  extent 
the  appearance  of  the  bacillus  of  malignant 
oedema,  but  with  this  difference,  that  the  ends 
of  the  Bacillus  fusiformis  are  not  square-cut 
but  rounded  or  tapering  which  gives  them  their 
characteristic  fusiform  appearance. 

The  number  of  the  bacilli  in  a  preparation 
depends  upon  the  severity  of  the  case  from 
which  it  is  derived :   if  it  be  a  mild  case  twenty 
or  thirty  may  be  found,  but  if  severe  the  number  is  so  considerable  as  to  be 
truly  described  as  a  pure  culture. 


of  hospital   gangrene   or 


576  ANAEROBIC   ORGANISMS   IN   GANGRENE 

Numerous  other  organisms  are  always  found  in  association  with  the  Bacillus 
fusiformis  in  these  lesions  (vide  ante). 

In  wounds  which  have  been  treated  with  antiseptics  many  involution  forms  are 
found :  vacuolated  bacilli  with  spindle-shaped  ends  or  indented  edges,  and  long 
forms  with  constrictions  which  stain  well  and  swellings  which  do  not  take  the 
stain. 

Staining  reactions. — The  Bacillus  fusiformis  is  easily  stained  by  the  basic 
aniline  dyes  and  best  by  carbol-fuchsin  or  one  of  the  carbol- violet  stains. 
The  bacillus  is  gram-negative. 

When  stained  with  methylene  blue  portions  of  the  organisms  do  not  take 
the  stain  :  these  unstained  areas  are  not  round  and  are  obviously  not  spores, 
as  they  do  not  stain  by  the  methods  used  for  staining  spores. 

Sections. — Fix  the  pieces  of  tissue  for  cutting  sections  in  a  saturated  aqueous 
solution  of  corrosive  sublimate,  and  harden  in  increasing  strengths  of  alcohol. 
Stain  in  carbol-thionin.  Vincent  recommends  the  following  technique  : 

1.  Stain  for  10  minutes  in  carbol-thionin. 

2.  Treat  for  a  few  seconds  with  the  following  solution  : 

Absolute  alcohol,  200        c.c. 

Iodine,        -  -       .  -  0*01  gram. 

3.  Pour  off  the  iodine  solution  and  treat  with  absolute  alcohol  or  alcohol 
tinted  with  safranin  or  fluorescin. 

4-  Clear  in  aniline  oil.     Wash  in  toluene. 

5.  Mount  in  balsam. 

In  sections  stained  by  this  method  two  layers  may  be  made  out : 

(a)  A  superficial  layer  1-3  mm.  thick,  stained  bluish  grey,  and  composed  of  a 
diphtheroid  exudate  remarkably  poor  in  cellular  elements  in  its  superficial  part  but 
in  its  deeper  layers  packed  with  bacilli.  Below  the  layer  of  bacilli  a  mass  of  leuco- 
cytes will  be  noticed. 

(6)  A  layer  composed  of  dead  tissue  from  which  all  trace  of  structure  has  dis- 
appeared for  part  of  its  thickness. 

Note,  (a)  There  are  certain  discrepancies  in  the  different  descriptions  of  the 
Bacillus  fusiformis.  By  the  majority  of  bacteriologists  the  bacillus  is  regarded 
as  non-motile  (Vincent,  Muhlens,  Ellermann,  Weaver  and  Tunnicliffe)  but  Letulle 
describes  it  as  motile  in  saliva,  and  according  to  Vespremy  it  is  provided  with 
numerous  flagella :  Plaut  moreover  describes  very  numerous,  very  delicate  flagella 
like  a  layer  of  cotton-wool  all  round  the  bacillus.  Nearly  all  observers  state  that 
the  bacillus  is  gram-negative  but  Plaut  is  of  a  contrary  opinion  and  is  supported  by 
Jungano  and  Distaso. 

(6)  The  variations  in  the  morphology  of  the  Bacillus  fusiformis  have  led  Ellermann 
to  describe  several  species  (three  types)  but  of  this  there  is  no  proof.  Similarly, 
there  is  no  reason  to  suppose  that  the  Bacillus  fusiformis  is  merely  a  cultivation 
form  of  a  spirochsete  as  Silberschmidt  has  suggested. 

4.  Cultural  characteristics. 

Vincent  had  always  failed  no  matter  what  media  he  used  to  obtain  pure 
cultures  of  the  Bacillus  fusiformis  from  cases  of  hospital  gangrene  or  Vincent's 
angina. 

If  a  portion  of  the  exudate  on  the  tonsil  were  sown  on  Martin's  broth  an  impure 
culture  was  obtained  in  which  the  bacillus  took  the  form  of  elongated,  non- motile 
filaments. 

More  recently  however  several  observers  utilizing  Veillon's  technique  have 
obtained  pure  cultures  of  the  bacillus.  It  is  a  strictly  anaerobic  organism, 
grows  only  at  37°  C.  and  in  media  to  which  serous  fluids  have  been  added  : 
sugar  appears  to  favour  its  growth.  Eichmeyer  lays  stress  on  the  importance 
of  keeping  the  material  at  a  constant  temperature  of  37°  C.  and  of  heating 
the  medium  to  that  temperature  before  sowing.  In  cultures  the  bacillus 


SPIRILLUM  NIGRUM  577 

remains  alive  for  20-25  days  :  it  produces  no  gas  but  gives  off  a  disagreeable 
smell. 

Isolation.  —  Lewkowicz  isolates  the  bacillus  in  Veillon's  tubes  containing 
glucose  agar  to  which  one-third  its  volume  of  the  peritoneal  fluid  of  a  child 
is  added.  After  incubating  at  37°  C.  for  4  days  sub-cultures  can  be  sown  in 
glucose-agar  or  glucose-broth  to  both  of  which  it  is  necessary  to  add  some 
serous  fluid. 

Ellermann  recommends  the  following  method  for  the  purpose  of  isolating 
the  bacillus  from  the  mouths  of  healthy  persons  : 

1.  Sow  some  dental  tartar  in  Cibil's  broth  and  incubate  at  37°  C.  :    after 
2  days  a  deposit  will  have  formed  consisting  of  cocci  and  the   Bacillus 
fusiformis. 

2.  At  the  same  time  sow  a  tube  of  sloped  agar  freely  with  dental  tartar  ; 
then  sow  a  tube  of  broth  with  the  growth  obtained  (staphylococci  and  strepto- 
cocci). 

3.  Sterilize  the  aerobic  broth  culture,  decant  the  broth  and  sow  it  anaerobi- 
cally  with  the  impure  culture  in  Cibil's  broth  :   the  medium  is  exhausted  for 
the  cocci,  and  the  fusiform  bacillus  grows  abundantly  in  pure  culture.     After 
sowing  a  few  sub-cultures  anaerobically  on  serum-agar  a  pure  culture  of  the 
Bacillus  fusiformis  is  obtained. 

Glucose-serum-agar.  (a)  Stab  culture.  —  Deep  stab  cultures  in  glucose- 
serum-agar  give  rise,  after  incubating  for  3  or  4  days,  to  a  minimal  growth 
consisting  of  a  greyish  streak  which  extends  to  within  1  cm.  of  the  surface 
of  the  agar  (Lewkowicz). 

(f3)  Isolated  colonies.  Veillon's  tubes.  —  After  incubating  for  24-48  hours 
small,  delicate,  opaque  colonies  appear,  greyish  or  yellowish-white  in  colour 
with  a  deeper  coloured  centre  :  on  further  incubation  (a  fortnight  or  3  weeks) 
the  colonies  may  attain  a  diameter  of  2  mm.  (Miihlens,  Ellermann). 

(7)  Plates.  —  When  sown  in  vacuo  on  the  surface  of  glucose-agar  containing 
an  albuminous  fluid  the  Bacillus  fusiformis  grows  as  small  grey  points,  trans- 
lucent and  thin,  which  may  reach  a  diameter  of  1'25  mm.  and  which  show 
under  the  microscope  a  very  delicate  festooned  margin  (Lewkowicz). 

Broth.  —  In  glucose-broth  containing  a  serous  fluid,  after  incubating  for  a 
few  days,  the  bacillus  produces  a  somewhat  abundant  greyish  white  deposit, 
the  broth  remaining  clear  (Lewkowicz). 


VIII.   SPIRILLUM  NIGRUM. 

The  Spirillum  nigrum  was  described  by  Rist  in 


suppurations  of  the  ear. 

Guinea-pigs  are  the  most  susceptible  animals,   ,  /     , 
but   the  pathogenic  properties  of  the   organism  \    >, 
for  the  lower  animals  are  very  slight. 

Microscopical  appearance.  —  The  Spirillum  ni-  _-^\o 

grum  is   a    small,    very  slender  organism  of  the 
shape  of  a  parenthesis  or  an  S  ;    its    ends   are  ^       /"* 

rounded  and  it  is  marked  by  a  small  black  point  FIG.  272.  —  Spirillum  nigrum. 
either  in  the  centre  or  at  one  of  the  ends.  It  is  gft,^  an  xagioooculture'  Car" 
highly  motile. 

Staining  reactions.  —  The  Spirillum  nigrum  stains  with  considerable  diffi- 
culty ;  the  only  satisfactory  dye  is  carbol-fuchsin  without  heat.  The  spirilla 
stain  bright  red  and  present  a  granular  appearance. 

The  organism  is  gram-negative. 

Cultural   characteristics.  —  The  Spirillum  nigrum  is  a   strictly  anaerobic 

2o 


578  ANAEROBIC  ORGANISMS  IN  GANGRENE 

organism  and  grows  on  the  ordinary  media  even  at  the  temperature  of  the 
laboratory.  Cultures  remain  alive  for  a  long  time  and  give  off  an  odour  of 
rotten  eggs. 

Colonies  on  agar  and  gelatin  are  characterized  by  their  intense  black 
colour  :  gelatin  is  not  liquefied. 

IX.  STAPHYLOCOCCUS  PARVULUS. 

This  organism  was  isolated  by  Veillon  and  Zuber  from  pus  from  a  case 
of  appendicitis  and  has  since  been  found  in  appendicitis,  in  cases  of  pulmonary 
gangrene  (Guillemot),  in  cases  of  infection  of  the  urinary  tract  (Cottet, 
Jungano),  and  in  the  mouths  of  very  young  children  (Micrococcus  gazogenes 
alcalescens  of  Lewkowicz). 

The  organism  is  only  slightly  pathogenic  for  laboratory  animals  ;  it  gives 
rise  to  an  abscess  when  inoculated  sub-cutaneously  into  rabbits  and  guinea- 
pigs  (Veillon  and  Rist). 

Microscopical  appearance. — The  Staphylococcus  parvulus  is  a  very  delicate 
coccus  smaller  than  the  Stapliylococcus  py(>genes  and  arranged  in  diplococci 
or  in  masses. 

Staining  reactions. — The  Stapliylococcus  parvulus  stains  feebly  with  methy- 
lene  blue  ;  the  best  stain  is  carbol-fuchsin.  It  is  gram -negative. 

Cultural  characteristics. — The  coccus  grows  anaerobically  on  all  media,  and 
at  the  ordinary  temperature  but  better  at  37°  C.  It  produces  a  little  gas. 
Broth  becomes  cloudy  and  a  dust-like  deposit  is  formed.  Cultures  on  agar 
and  gelatin  are  in  no  way  characteristic.  Gelatin  is  not  liquefied. 


X.   MICROCOCCUS  FETIDUS. 

This  organism  was  found  by  Veillon  in  pus  from  cases  ot  gangrene,  by  Rist 
in  cases  of  suppuration  of  the  ear,  by  Guillemot  and  Cottet  in  cases  of  pul- 
monary gangrene,  by  Jeannin  in  cases  of  putrid  puerperal  infection,  and  by 
Halle  in  the  vagina,  in  pus  from  Bartholin's  glands,  etc. 

It  is  pathogenic  for  rabbits  and  especially  for  guinea-pigs  which  succumb 
in  a  few  days  after  sub-cutaneous  inoculation. 

Microscopical  appearance. — The  organism  occurs  as  single  cocci  or  as  cocci 
arranged  as  diplococci,  and  occasionally  it  forms  small  masses.  Tn  cultures 
the  diplococci  are  arranged  in  very  short  chains  of  three  or  four  diplococci. 

Staining  reactions. — The  Micrococcus  fetidus  stains  easily  with  the  basic 
aniline  dyes  and  is  gram-positive. 

Cultural  characteristics. — The  Micrococcus  fetidus  grows  anaerobically  on 
all  the  ordinary  media  between  22°  and  37°  C.  :  at  22°  C.  the  growth  is  poor 
and  small  in  amount.  In  artificial  culture  it  produces  very  fetid  smelling 
gases.  In  broth  it  gives  rise  to  an  uniform  turbidity  ;  isolated  colonies  on 
agar  are  small  and  round  and  are  not  particularly  characteristic.  Growth 
on  gelatin  at  22°  C.  is  slow  and  scanty  ;  the  medium  is  not  liquefied. 


XI.   BACILLUS  AEROBICUS  SEPIS. 

Legros  has  recorded  two  cases  of  gaseous  gangrene  in  man  which  were  caused  by 
an  aerobic  bacillus  to  which  he  has  given  the  name  Bacille  septique  aerobic.  Haute" 
found  the  same  organism  associated  with  staphylococci  and  streptococci  in  a  case 
of  puerperal  infection  and  also  in  a  case  of  appendicitis. 

The  bacillus  is  a  rod- shaped  organism  with  rounded  ends,  motile,  straight  or 
slightly  curved  and  measuring  about  3/x  x  0'5-1/j-,  and  sometimes  occurs  as  short 


BACILLUS  AEROBICUS  SEPIS  579 


A* 


chains.     It  forms  spores.     The  bacillus  stains  with  the  basic  aniline  dyes  and  is 
gram-positive. 

Though  principally  aerobic  it  can  nevertheless  grow  under  anaerobic  conditions : 
the  temperature  of   growth  lies  between    18°   and 
43°  C. 

Broth  cultures  give  off  a  most  offensive  butyric 
odour. 

Gelatin  is  rapidly  liquefied.     The  bacillus  grows      f  &P-      I        "iT 
exceedingly  well  on  agar  and  potato.     It  does  not  f\     f^Jt. 

produce  indol,  and  coagulates  milk  in  fine  granular  .    \A 

flakes  without  altering  the  reaction  of  the  medium.  •!  %L 

It  slowly  liquefies  coagulated  serum. 

Inoculated  sub-cutaneously  into  guinea-pigs  it  pro-  JF  N  IB* 

duces  sometimes  a  gaseous  gangrene  with  a  sub- 
normal temperature  which  terminates  fatally  (adult  t»      %? 
animals),  at  other  times  a  septicaemia  without  local  \| 
lesions  (young  animals    and   pregnant   does).     The 

virulence  of  the  organism  is  maintained  with  dim"-    FlG  273  _ Bacillus  aerobicus  sepis. 
culty  :    it  can  be  increased  by  inoculating  a  little  (After  Legros.) 

lactic  acid  with  the  culture. 

The  experiments  of  Rosenthal  have  shown  that  it  is  easy  to  modify  the  conditions 
of  the  growth  of  the  bacillus  and  to  vary  the  capacity  of  one  and  the  same  strain 
for  living  in  oxygen- containing  media  or  in  those  from  which  air  is  removed.  It 
is  unnecessary  perhaps  to  point  out  that  it  is  under  anaerobic  conditions  that  the 
bacillus  sets  up  the  lesions  of  gaseous  gangrene. 


CHAPTER  XL. 
THE   PNEUMOCOCCUS. 

Synonyms :  Streptococcus  lanceolatus  :  Micrococcvs  pasteuri. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  581. 

Section  II. — Morphology,  p.  582. 

1.  Microscopical  appearance  and  staining  reactions,  p.  582.  2.  Cultural  charac- 
teristics, p.  584. 

Section  III. — Biological  properties,  p.  585. 

1.  Vitality  and  virulence,  p.  585.  2.  Bio-chemical  reactions,  p.  586.  3.  Toxins, 
p.  586.  4.  Vaccination,  p.  587.  5.  Serum  therapy,  p.  588.  6.  Agglutination, 
p.  589.  7.  Precipitins,  p.  590.  8.  Immune  body,  p.  590. 

Section  IV. — Detection.,  isolation  and  identification  of  the  pneumococcus,  p.  590. 

THE  pneumococcus  is  the  infecting  agent  in  acute  lobar  pneumonia  (Talamon, 
Frankel),  but  this  does  not  represent  the  limit  of  its  setiological  role  for  it  is 
concerned  in  by  far  the  greater  number  of  the  complications  of  pneumonia, 
as  well  as  in  certain  other  affections. 

(i)  The  pneumococcus  is  frequently  found  in  the  saliva  of  healthy  persons  (Pasteur 
and  others).  Netter  found  it  in  the  saliva  of  persons  who  had  recently  recovered 
from  an  attack  of  pneumonia  in  four  out  of  five  cases  examined,  and  once  out  of  five 
cases  examined  in  that  of  persons  who  had  never  suffered  from  the  disease.  He 
showed  that  during  the  early  stages  of  the  disease  the  pneumococcus  in  the  saliva  of 
the  persons  included  in  the  former  series  was  virulent,  that  it  disappeared  at  the 
time  of  the  crisis,  and  reappeared  at  the  end  of  a  fortnight.  The  pneumococcus 
is  an  habitual  inhabitant  of  the  tonsillar  mucus  (Bezan9on  and  Griffon),  and  Burger 
isolated  it  from  the  mouths  of  many  healthy  persons  (34  out  of  100  examined).  In 
the  latter  it  lives  as  a  harmless  parasite  in  the  mouth :  but  should  the  resistance  of 
the  tissues  from  any  cause  become  lowered,  the  organism  overcomes  the  leucocytic 
defences  and  invades  the  lung. 

(ii)  In  lobar  pneumonia,  the  pneumococcus  is  always  found  in  the  area  of  hepatiza- 
tion  where  it  may  be  present  in  pure  culture  or  be  associated  with  other  micro- 
organisms, generally  streptococci,  staphylococci  and  the  bacillus  of  Friedlander : 
it  occurs  also  in  the  characteristic  rusty  sputum.  Some  cases  of  broncho-pneumonia 
are  due  to  the  pneumococcus. 

(iii)  The  pneumococcus  occasionally  passes  into  the  blood- stream  and  gives  rise 
to  complications,  often  of  the  nature  of  a  suppuration,  in  the  neighbourhood  of  the 
lung  or  in  more  distant  parts  (Friedlander,  Talamon,  Frankel).  Pneumococcal  pus 
is  thick  and  viscous,  very  rich  in  cellular  elements  and  greenish  in  colour.  Sup- 
puration due  to  the  pneumococcus  tends  to  undergo  spontaneous  resolution. 

(iv)  Pleurisy,  pericarditis — fibrinous  or  purulent,  endocarditis — vegetative  or 
ulcerative,  meningitis,  nephritis,  suppurative  parotitis,  suppurative  arthritis, 
peritonitis,  metritis  and  abscesses,  as  complications  of  pneumonia  may  any  or  all 


EXPERIMENTAL  INOCULATION  581 

of  them  be  due  to  the  pneumococcus,  though  it  is  to  be  remembered  that  they  may 
be  caused  equally  by  any  of  the  other  organisms  of  suppuration. 

(v)  Over  and  above  such  cases  as  these  in  which  pneumonia  is  a  co-existent 
symptom,  the  pneumococcus  may  also  be  the  primary  cause  of  fibrino-purulent 
pleurisy,  sero-fibrinous  or  suppurative  pericarditis  (Osier,  Banti),  conjunctivitis, 
keratitis,  suppurative  otitis  (Zaufal,  Netter),  ulcerative  endocarditis  (Jaccoud  and 
Netter,  Weichselbaum),  simple  or  membranous  inflammation  of  the  throat  (Cornil, 
Jaccoud,  Menetrier,  Rendu  and  Baulloche),  peritonitis,  and  suppuration  of  the 
biliary  passages. 

(vi)  The  pneumococcus  is  also  the  primary  cause  of  a  large  number  of  cases  of 
meningitis,  frequently  of  the  cerebro-spinal  type :  Netter  found  this  organism  in 
eighteen  out  of  thirty- one  cases  of  meningitis  neither  accompanied  nor  followed  by 
pneumonia. 

The  pneumococcus  is  the  cause  of  a  small  minority  of  cases  of  epidemic  cerebro- 
spinal  meningitis  (Foa,  Landouzy)  though  it  is  now  well  known  that  epidemic 
cerebro-spinal  meningitis  is  usually  due  to  the  Meningococcus  (q.v.). 

Marchoux  has  described  an  epidemic  of  cerebro-spinal  meningitis  among  the 
natives  of  Senegal  which  occurred  coincidently  with  numerous  cases  of  pneumonia 
and  which  was  caused  by  the  pneumococcus. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

Mice  are  the  most  susceptible  animals  :  then  in  order  of  decreasing  suscepti- 
bility come  rabbits,  rats,  sheep,  guinea-pigs  and  dogs.  Pigeons  are  immune. 

Mice. — Sub-cutaneous  inoculation  of  a  small  quantity  of  a  culture  of  the 
pneumococcus  or  of  a  pneumococcal  exudate  invariably  leads  to  the  death 
of  the  animal  in  12-30  hours.  Mice  succumb  as  the  result  of  a  pneumococcal 
septicaemia  without  showing  any  pulmonary  symptoms  :  at  the  site  of  inocula- 
tion only  is  there  a  little  oedema.  Post  mortem,  there  is  no  lesion  other  than 
an  enlargement  of  the  spleen  :  the  blood  is  black  and  contains  a  large  number 
of  encapsulated  pneumococci,  as  do  also  the  spleen  and  other  internal  viscera, 
the  peritoneum  and  bone  marrow. 

Rabbits. — The  inoculation  of  rabbits  may  be  productive  of  either  one  of 
two  types  of  disease  according  to  the  virulence  of  the  organism  inoculated. 

(a)  Inoculation  of  a  virulent  virus. — Sub-cutaneous,  intra-peritoneal   or 
intra-venous  inoculation  leads  to  the  death  of  the  animal  from  septicaemia 
in  24-72  hours.     There  is  only  a  slight  local  reaction.     Post  mortem  the  spleen  is 
enlarged  and  the  pneumococcus  may  be  found  in  the  blood  and  internal  organs. 

Lobar  pneumonia  often  accompanied  by  pleurisy  on  the  same  side  follows 
inoculation  of  material  into  the  lung. 

(b)  Inoculation  with  an  attenuated  virus. — Sub-cutaneous  inoculation  proves 
fatal  though  much  less  rapidly  than  in  the  former  case.     There  is  an  inflam- 
matory reaction  at  the  site  of  inoculation  and  the  animal  dies,  not  from  a 
septicaemia  without  visceral  localization,  but  from  a  true  lobar  pneumonia 
frequently  accompanied  by  pleurisy,  pericarditis,  peritonitis,  arthritis,  etc. 

Rats. — Rats  only  succumb  after  the  inoculation  of  very  much  larger  quan- 
tities of  the  virus  than  are  necessary  to  kill  mice  and  rabbits.  There  is  an 
intense  inflammatory  reaction  at  the  site  of  sub-cutaneous  inoculation,  and 
the  oedema  may  extend  over  the  whole  of  the  abdominal  and  thoracic  walls  : 
lobar  pneumonia  not  infrequently  results.  Post  mortem  only  a  few  pneumo- 
cocci are  found  in  the  blood.  Intra-pulmonary  inoculation  produces  a  patch 
of  lobar  pneumonia  accompanied  by  a  sero-fibrinous  pleurisy. 

Sheep. — To  produce  a  fatal  infection  in  sheep  by  sub-cutaneous  inoculation 
more  than  1  c.c.  of  culture  must  be  used.  An  extensive  oedema  develops 
around  the  site  of  inoculation  :  when  death  takes  place  only  a  few  micro- 
organisms are  found  in  the  blood. 

Intra-pulmonary  inoculation  is  followed  by  a  fatal  attack  of  pneumonia. 


582  THE   PNEUMOCOCCUS 

Intra-tracheal  inoculation  appears  to  do  no  harm,  though  Gamaleia  after 
irritating  the  respiratory  passages  by  the  injection  of  a  solution  of  tartarated 
antimony  produced  a  fatal  pneumonia  by  this  method. 

The  vitality  and  virulence  of  the  pneumococcus  become  rapidly  attenuated 
by  passage  through  sheep  so  that  serial  inoculations  are  impossible. 

Guinea-pigs. — Guinea-pigs  are  comparatively  immune  to  the  pneumococcus. 
Sub-cutaneous  inoculation  is  followed  by  a  more  or  less  marked  local  reaction 
which  very  often  spontaneously  resolves.  Intra-peritoneal  inoculation  is 
more  severe  and  is  often  followed  by  death. 

Dogs. — Dogs  only  succumb  to  extremely  large  doses.  A  very  extensive 
oedema  is  produced  as  the  result  of  sub-cutaneous  inoculation,  and  on  rare 
occasions  death  may  take  place  about  the  fourth  or  fifth  day  :  the  blood 
will  contain  a  very  few  pneumococci. 

Intra-pulmonary  inoculation  sets  up  a  pneumonia  which  runs  the  same 
course  as  in  man,  but  which  as  a  rule  resolves. 

Intra-tracheal  inoculation  is  generally  without  effect :  Tchistovich  however 
produced  a  fatal  result  in  3  out  of  19  dogs  inoculated  by  this  method.  In 
performing  the  experiment  great  care  must  be  taken  that  the  tissues  around 
the  trachea  are  not  infected  (for  technique,  see  p.  179),  otherwise  the  result 
will  be  misleading. 

To  summarize.  The  most  susceptible  animals  die  from  a  pneumococcal 
septiccemia.  Pneumonia  occurs  more  often  in  the  less  highly  susceptible  animals. 
Further,  in  accordance  with  the  general  ride,  the  severity  of  the  infection  is  in 
inverse  ratio  to  the  extent  of  the  local  lesion. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance  and  staining-  reactions. 

The  pneumococcus  presents  under  the  microscope  two  different  appear- 
ances according  as  to  whether  it  has  been  obtained  from  human  or  animal 
tissues  or  from  cultures  on  artificial  media.  In  cultures  on  liquid  albuminous 
media  (serum,  or  broth  containing  fresh  blood,  etc.)  the  pneumococcus  has 
however  the  same  characteristics  as  in  the  tissues. 

A.  Appearance  in  the  tissues. — The  pneumococcus  in  sputum,  blood, 
scrapings  of  organs,  etc.  occurs  as  a  coccus,  sometimes  rounded  but  generally 

^X 

k 


t^^g? 
fe^ 


FIG.  -274.— Pneumococcus   in   sputum.     Gram's   stain   and   dilute   carbol- 
fuchsin.     (Oc.  2,  obj.  TUh,  Zeiss.) 


MORPHOLOGY  583 

oval,  and  slightly  pointed  at  the  ends  (hence  the  comparison  with  grains  of 
barley  or  a  candle  flame  and  the  synonym  Diplococcus  lanceolatus) .     Two 
cocci   are   generally   joined   together— with   their   long   axes    in    the    same 
straight  line,  like  a  figure  of  8 — forming  a  diplococcus,  but  here  and  there  a 
few  single  cocci  may  be  found,  and  sometimes  short  chains  composed  of 
three  or  four  cocci.     The  organisms  whether  occurring  as  single  cocp.i,  diplo- 
cocci  or  in  chains  are  surrounded  by  a  capsule 
or  areola,  which  is  a  sort  of  albuminous  en-          S."^ 
velope :   this  capsule  can  be  stained  by  appro-  *  9 

priate  methods.  % 

The  pneumococcus  varies  considerably  in  size :          %% 
0-5orO'75/xtolorl-25/*.  * 

Staining    reactions. — The    pneumococcus    is       4jSa-  * 
readily  stained  with  the  basic  aniline  dyes  and        $KJs 
is  gram-positive.     The  following  methods  are  ^ 

recommended  for  diagnostic  purposes.  jf  ^     **'•>.<, 

(a)  Nicolle's  method.  Recommended. — Films 
prepared  in  the  ordinary  way  are  stained  with 
carbol-thionin  for  some  seconds  then  passed 

•  i  i     Ai  TIT.!  /-i  j_     o\  i      i       FIG.  27o. — Pneumococcus.     x  1000. 

quickly  through  alcohol-acetone  (1  to  3),  washed,  stained  to  show  capsules, 

dried  and  mounted. 

(6)  Gram's  method. — Gram's  method  should  always  be  used  in  the  identifi- 
cation of  the  pneumococcus  ;  blood-films  give  very  pretty  preparations. 
The  double  staining  method  recommended  on  p.  207  should  be  adopted.  The 
capsules,  as  a  rule,  remain  unstained. 

(c)  Capsule  staining. — The  method  (a)  recommended  above  for  staining 
the  pneumococcus  will  stain  the  capsules  :  several  other  methods  of  capsule 
staining  applicable  to  the  pneumococcus  have  already  been  described  in  the 
earlier  part  of  the  book  (p.  147). 


*-'*       v     1"      9       ^ 

4;  \V$j; 

*       tjw  >    %       **£$  ;:      •^ 

^       ^V  '        -  ^         "' 

*  **  ':'^*r  :^Sg<  ^c  ^  "s 


FIG.    276. — Pnenmoc.occus.     Section    of    lung.     Gram's    stain    and    eosin. 
(Oc.  2,  obj.  TUh,  Zeiss.) 

(d)  Section  staining. —  (i)  Sections  are  best  stained  by  Gram's  double  or 
triple  stain  (p.  219).  Weigert's  method  may  also  be  used  (p.  216). 

(ii)  It  is  a  somewhat  difficult  matter  to  stain  capsules  in  sections  :  one  or 
other  of  the  following  methods  may  be  tried. 


584 


THE  PNEUMOCOCCUS 


Friedlander's  method.  —  1.  Stain  the  section  in  the  following  solution  for 
24  hours  : 

Fuchsin,      -  1  gram. 

Absolute  alcohol,  5  grams. 

Glacial  acetic  acid,        -  2       ,, 

Distilled  water,    -  100      „ 

2.  Wash  in  alcohol  and  then  in  2  per  cent,  acetic  acid  for  2  minutes. 

3.  Wash  in  distilled  water,  dehydrate  in  absolute  alcohol,  clear  in  clove 
oil  or  xylol  and  mount  in  balsam. 

Ribbert's  method.  —  1.  Stain  the  section  for  a  few  minutes  in  the  following 
solution  : 


Distilled  water,    - 
95  per  cent,  alcohol, 
Glacial  acetic  acid, 
Violet-dahlia, 


grams. 


2. 


-  100 
50 
12*5  „ 

Q.8.  to  saturate 
in  the  warm. 

Wash  in  water,  dehydrate  in  absolute  alcohol,  clear  in  clove  oil  or  xylol. 


and  mount  in  balsam. 

B.  Appearance  in  cultures.  —  In  cultures  on  artificial  media  generally  the 
pneumococcus  is  not  encapsulated  ;  capsules  are  only  found  in  cultures  in 
liquid  serum  or  blood-broth. 

In  cultures,  the  pneumococcus  appears  either  as  lancet-shaped  cocci,  or  as 
rounded  grains  :  the  latter  may  be  found  to  the  absolute  exclusion  of  the 
lanceolate  forms.  The  organism  may  occur  as  single  cocci,  as  diplococci, 
or  in  short  streptococcal  chains  of  3  to  8  elements  :  the  chains  consist  of 
chains  of  diplococci,  the  long  axes  of  the  cocci  being  dis- 
posed along  the  line  of  the  chains.  The  latter  are  especially 
numerous  and  long  in  broth  cultures. 

2.  Cultural  characteristics. 

Conditions  of  growth.  —  The  pneumococcus  is  a  facultative 
aerobe.  Growth  does  not  take  place  below  25°  C.  so  that 
the  organism  cannot  be  cultivated  on  ordinary  gelatin.  The 
optimum  temperature  is  about  35°-37°  C..  and  development 
ceases  at  42°  C.  Growth  is  more  vigorous  in  liquid  than  on 
solid  media,  and  the  medium  must  be  faintly  alkaline  : 
ordinary  media  are  not  very  suitable  for  cultivating  the 
pneumococcus. 

Agar.  —  After  incubating  for  24  hours  at  37°  C 
growth  of  small  transparent  colonies  resemblin 
dew  is  seen.  The  colonies  are  difficult  to  see  am 
confluent. 

Coagulated  serum.  —  The  growth  is  similar  to  that  on  agar, 
but  differs  from  the  latter  in  that  the  colonies  occasionally 
coalesce  and  form  a  thin  semi-transparent  film. 

Burger's  serum  (p.  590). 

Broth.—  After  incubating  at  37°  C.  for  24  or  36  hours  the 
medium  is  very  slightly  cloudy  :  a  very  delicate  powdery 
deposit  is  precipitated  later.  Broth  containing  8  per  cent. 
glucose  is  a  much  better  medium  than  ordinary  broth 
(Turro). 

Broth  containing  rabbit's  blood.—  To  prepare  this  medium  a  little  blood  is 
collected  aseptically  from  the  ear  vein  of  the  rabbit  (p.  194)  and  added  to 
sterilized  broth  in  the  proportion  of  1  part  of  blood  to  3  or  4  parts  o 


a  delicate 
drops  of 
are  never 


FIG.  277.— Pneu- 
mococcus. Surface 
culture  on  agar  (3 
days  at  37°  C.). 


BIOLOGICAL  PROPERTIES  585 

broth.  In  this  medium  the  pneumococcus  grows  abundantly  at  37°  C.  pro- 
ducing a  marked  cloudiness  of  the  medium  and  later  a  mucoid  precipitate 
very  rich  in  micro-organisms. 

Liquid  serum. — The  best  serum  is  that  prepared  with  young  rabbit  blood 
collected  aseptically  and  not  heated  :  in  such  a  serum  a  very  copious  growth 
is  obtained  on  incubating  at  37°  C.  At  first  the  medium  is  thickened  and 
becomes  markedly  turbid,  and  later  a  heavy  precipitate  composed  of  capsulated 
pneumococci  is  thrown  down. 

Milk. — The  pneumococcus  coagulates  milk  [but  not  invariably,  see  p.  602]. 

Potato. — No  growth  takes  place  on  this  medium. 

Inulin  media. — American  observers  recommend  media  containing  inulin 
for  isolating  and  differentiating  the  pneumococcus.  The  pneumococcus  does 
not  ferment  inulin,  herein  presenting  a  contrast  to  the  streptococci  (cf.  p.  602). 

Hiss  uses  the  following  medium :  To  1  part  of  ox  serum  add  2  parts  of  distilled 
water  and  1  part  per  cent,  of  litmus  solution.  Heat  to  100°  C.,  add  1  per  cent,  of 
inulin  and  sterilize  on  three  successive  days  at  a  low  temperature. 

Ruediger  recommends  the  following  medium  for  the  isolation  of  the  pneumo- 
coccus :  to  1  litre  of  broth  add  15  grams  of  agar  and  15  grams  of  inulin  and,  after 
sterilization,  20  c.c.  of  a  5  per  cent,  solution  of  Merck's  litmus.  Distribute  in  tubes 
and  to  each  tube  add  1  c.c.  of  ascitic  fluid. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Vitality  and  virulence. 

I.  In  sputum  and  in  albuminous  exudates  the  pneumococcus  retains  its 
vitality  and  virulence  for  a  long  time  and  can  resist  even  prolonged  desicca- 
tion (Bordoni)  :  it  also  shows  prolonged  vitality  in  the  soil  and  in  dust.     Em- 
merich found  virulent  pneumococci  in  the  dust  between  the  joists  in  wards 
where  pneumonia  patients  were  lying,  and  Uffelmann  discovered  the  organism 
in  the  air  of  a  vault. 

II.  In  artificial  cultivation  the  pneumococcus  soon  loses  its  virulence  and 
even  its  vitality.     Cultures  on  solidified  serum  and  on  agar  die  after  4  or  5 
days  ;   cultures  in  liquid  media  remain  alive  longer,  but  are  avirulent  at  the 
end  of  a  week.     The  less  suited  the  medium  is  to  the  growth  of  the  pneumo- 
coccus the  more  rapidly  does  the  virulence  diminish  ;  thus  it  disappears  more 
quickly  in  ordinary  broth  than  in  blood-broth.     According  to  Bezan9on  and 
Griffon,  the  pneumococcus  will  live  for  a  year  in  defibrinated  blood  containing 
ascitic  fluid.     (In  this  medium  the  pneumococcus  appears  as  chains :  sown 
afterwards  on  agar  or  broth  it  preserves  this  inherited  appearance  but  reverts 
to  the  diplococcal  form  in  rabbit-serum.)     Defibrinated  rabbit  or  dog  blood 
either  pure  (Gilbert  and  Fournier)  or,  better  still,  blood-agar  (Bezangon  and 
Griffon)  may  also  be  used  as  media  on  which  to  preserve  the  vitality  of  the 
organism.     In  ordinary  media  the  virulence  rapidly  diminishes  and  is  alto- 
gether lost  in  the  third  sub-culture. 

Cultures  are  sterilized  in  24  hours  at  a  temperature  of  42°  C.  ;  in  10  minutes 
at  56°  C.  and  instantly  at  65°-70°  C.  Desiccation  rapidly  kills  the  organism 
in  culture. 

Pasteur  showed  that  the  attenuation  in  culture  is  largely  due  to  the  action  of 
the  oxygen  of  the  air ;  hence  it  follows  that  the  virulence  can  be  maintained  for  a 
long  time  in  anaerobic  culture  (Frankel).  In  cultures  on  egg  sown  as  described  on 
p.  53  (A)  the  organism  will  remain  virulent  for  several  months  (Bunzl-Federn).  An 
efficient  method  of  preserving  the  virulence  of  the  pneumococcus  is  to  inoculate  the 
culture  into  a  rabbit,  collect  a  little  of  the  heart- blood  post  mortem,  and  store 
it  in  a  sealed  pipette :  in  the  pipette  the  blood  retains  its  virulence  for  a  very  long 
time.  For  future  experiments  sow  the  blood  in  broth,  incubate  for  24^36  hours 
and  inoculate  the  culture. 


586  THE   PNEUMOCOCCUS 

Another  cause  of  the  attenuation  and  death  of  the  pneumococcus  in  culture  is 
the  rapidity  and  extent  to  which  it  forms  acid  (for  the  most  part  formic  acid).  The 
addition  of  calcium  carbonate  to  the  medium  neutralizes  the  acid  as  it  is  formed, 
and  in  this  way  the  vitality  of  the  organism  can  be  preserved  for  more  than  a  month 
(Wurtz  and  Mosny).  According  to  Bolduan,  the  calcium  salt  maintains  the  life 
of  the  culture  not  because  it  neutralizes  the  acid  but  on  account  of  a  special  action 
of  the  calcium  on  the  pneumococcus. 

Restitution  and  exaltation  of  virulence.— (a)  The  virulence  of  an  attenuated 
pneumococcus  can  be  restored  by  inoculating  into  a  rabbit  fairly  large  doses 
(1  c.c.)  of  a  broth  culture  of  the  organism  and  at  the  same  time  an  equal 
quantity  of  a  filtered  culture  of  Proteus  vulgaris.  The  animal  will  die  from  a 
pneumococcal  septicaemia  and  the  organism  in  the  blood  will  be  found  to  be 
virulent. 

(6)  The  virulence  of  the  pneumococcus  can  be  increased  by  passages  through 
rabbits.  Intra-venous  inoculation  is  better  for  this  purpose  than  sub- 
cutaneous, but  intra -peritoneal  inoculation  is  a  more  certain  method  than 
either  (Issaeff). 

Inoculate  1  or  1*5  c.c.  of  the  blood  of  a  rabbit  dead  of  a  pneumococcal  septicaemia 
into  the  peritoneum  of  a  rabbit  (A).  Inoculate  a  second  rabbit  with  the  blood  of 
A  and  so  on  in  series  until  eight  or  nine  rabbits  have  been  inoculated.  The  amount 
of  blood  inoculated  is  at  this  stage  diminished ;  for  the  eleventh  rabbit,  for 
instance,  6  or  8  drops  of  the  virus  are  sufficient. 

From  about  the  twelfth  passage  onward  the  blood  loses  its  power  of  coagulation, 
becomes  extremely  toxic  and  virulent  and  contains  very  numerous  pneumococci. 
One  drop  of  this  blood  inoculated  into  the  peritoneum  of  a  rabbit  will  prove  fatal 
in  10  or  12  hours :  if  too  large  a  dose  (for  example  1—2  c.c.)  be  given  the  rabbit  will 
die  very  quickly  (5  or  6  hours)  of  toxaemia  and  not  of  septicaemia. 

Sub-cutaneous  inoculation  of  4  or  6  drops  of  blood  containing  a  pneumo- 
coccus the  virulence  of  which  has  been  increased  in  this  way  will  kill  rabbits 
in  12  or  15  hours.  It  is  to  be  noted  however  that  after  a  long  series  of  passages 
through  rabbits  the  virulence  of  the  organism  becomes  attenuated  but  can 
be  restored  by  two  or  three  passages  through  another  species  such  as  the 
guinea-pig  or  dog. 

2.  Bio-chemical  reactions, 

The  bio-chemical  reactions  of  the  pneumococcus  and  the  characteristics 
which  distinguish  this  organism  from  the  streptococci  are  described  on 
p.  602. 

3.  Toxins. 

(i)  Filtered  cultures  of  the  pneumococcus  are  only  slightly  toxic.  If 
inoculated  in  large  quantities  into  the  veins  of  rabbits  they  give  rise  to  a 
transitory  rise  of  temperature  and  loss  of  weight :  death  does  not  generally 
result. 

A  somewhat  more  toxic  product  is  obtained  by  sterilizing  cultures  of  the 
organism  by  chloroform  or  heat  (a  temperature  of  58°  C.  for  2  hours  will 
sterilize  the  culture  without  altering  the  toxin).  Cultures  on  young  rabbit- 
serum  yield  the  most  powerful  toxin. 

Anaerobic  cultures  in  broth  or  serum  which  are  kept  alkaline  offer  no 
advantage  from  the  point  of  view  of  toxin  production. 

The  brothers  Klemperer  isolated  a  toxin  by  precipitating  filtered  broth  cultures 
with  alcohol  or  sulphate  of  ammonia  :  Foa  and  Carbone  obtained  similar  results. 
Andreini  attributed  the  toxicity  of  cultures  to  an  alkaloidal  base. 

(ii)  Emmerich  obtained  a  more  toxic  product  by  crushing  and  expressing 
the  organs  of  rabbits  which  had  died  of  a  pneumococcal  septicaemia  and 
filtering  the  juice  through  a  bougie.  Mosny  modified  this  method  as  follows  : 

Immediately  after  death  the  organs  of  the  rabbit  are  minced  and  macerated  in 


VACCINATION  587 

twice  their  weight  of  water  for  24  hours,  a  few  pieces  of  thymol  being  added  as  an 
antiseptic.  The  fluid  is  then  filtered  several  times  through  paper  and  finally  through 
a  Chamberland  bougie. 

(iii)  From  the  blood  of  rabbits  killed  by  his  virulent  virus  (vide  ante)  Issaeff 
obtained  a  toxin  capable  of  killing  rabbits  on  intra-venous  inoculation  when 
administered  in  a  dose  equivalent  to  one  one-hundredth  of  the  weight  of  the 
animal.  The  toxicity  of  the  product  is  considerably  diminished  by  heating  it 
to  70°  C.,  and  is  destroyed  at  100°  C.  His  method  is  as  follows  :— 

1.  Collect  under  aseptic  precautions  the  heart- blood  of  three  or  four  rabbits 
which  have  recently  succumbed  to  the  inoculation  of  a  virulent  pneumococcus 
(80-100  grams  of  blood)  and  mix  the  various  samples  in  a  sterile  vessel. 

2.  Add  an  equal  volume  of  sterile  water  containing  1  per  cent,  of  glycerin  and 
5  or  6  drops  of  a  saturated  solution  of  bicarbonate  of  sodium  per  100  c.c.     Mix. 

3.  Filter  the  mixture  through  a  Chamberland  bougie. 

The  toxicity  of  the  product  is  much  diminished  by  heating  to  70°  C.  and  destroyed 
at  100°  C. 

By  filtering  pleural  and  peritoneal  exudates  of  rabbits  which  had  died  after 
the  inoculation  of  a  virus  of  increased  virulence  Issaeff  was  also  able  to  obtain 
a  product  sufficiently  toxic  to  kill  rabbits. 

4.  Vaccination. 

(i)  With  toxins. — It  is  possible  to  immunize  an  animal  against  the  pneumo- 
coccus by  inoculating  it  with  filtered  cultures  or  with  toxins  prepared  by 
the  methods  of  Emmerich,  Mosny  or  IssaefT ;  the  immunity  however  is  of 
short  duration,  and  to  render  the  animal  more  permanently  immune  it  must 
be  inoculated  afterwards  with  living  cultures. 

A.  Heat  the  serum  of  rabbits  which  have  died  of  a  pneumococcal  infection  to 
58°  C.  and  inoculate  it  in  doses  of  10—20  c.c.  into  the  ear  vein  of  a  fresh  rabbit.     After 
four  or  five  inoculations  at  intervals  of  a  few  days  the  animal  is  able  to  resist  the 
inoculation  of  virulent  cultures  (Foa). 

B.  A  similar  result  is  obtained  by  inoculating  in  the  same  way  toxins  prepared 
by  the  methods  of  Emmerich  or  Mosny. 

C.  Issaeff  immunizes  rabbits  by  inoculating  into  the  veins  at  intervals  doses 
of  10-50  c.c.  of  sterilized  cultures  (broth  or  serum).     Each  inoculation  is  followed 
by  a  fairly  sharp  reaction  so  that  the  next  inoculation  must  be  withheld  until  the 
animal  appears  to  have  completely  recovered  from  the  previous  experiment. 

The  same  observer  has  also  immunized  rabbits  by  inoculating  them  with  toxins 
extracted  from  the  blood  as  described  above.  A  single  injection  of  10  c.c.  of  toxin 
into  the  blood  or  peritoneal  cavity  is  sufficient  to  render  rabbits  highly  immune 
against  the  pneumoccocus. 

The  immunized  animals  are  tested  by  inoculating  them  sub-cutaneously  with  the 
blood  of  a  rabbit  just  dead  of  a  pneumococcal  infection  ;  on  the  first  occasion  2-4 
drops  and  on  the  second  0*5  c.c.  of  the  blood  are  administered.  To  keep  up  the 
immunity  the  animal  should  be  re-inoculated  once  a  month  with  a  dose  of  not  more 
than  0'5  c.c.  sub-cutaneously.  Before  giving  the  test  inoculation  it  is  necessary 
to  wait  until  the  animal  has  completely  recovered  from  the  effects  of  the  immuniza- 
tion and  until  the  weight  has  begun  to  increase. 

D.  Neufeld  and  Handel  immunized  horses  by  inoculating  them  intra-venously 
with  organisms  killed  by  heat.     Highly  virulent  cultures  in  broth  were  heated  to 
60°  C.  and  centrifuged.     The  organisms  alone  were  inoculated  :  the  animals  tolerated 
the  inoculations  very  well  even  when  they  were  repeated  in  very  large  doses  at  fre- 
quent intervals.     The  resulting  serum  was  rich  in  thermostable  substances  which 
assisted  the  phagocytosis  of  virulent  pneumococci ;  a  dose  of  2  c.c.  intra-peritoneally 
immunized  mice  against  the  inoculation  of  0*1  c.c.  of  a  very  virulent  culture. 

Rabbits  vaccinated  with  toxin  are  absolutely  immune  to  infection  with 
living  cultures,  but  not  to  toxins,  to  which  they  react  even  more  violently 
than  do  normal  rabbits. 


588  THE   PNEUMOCOCCUS 

(ii)  With  attenuated  cultures. — Animals  can  be  immunized  by  inoculating 
them  with  living  cultures  the  virulence  of  which  is  attenuated  by  age.  Thus, 
for  example,  broth  cultures  5  or  6  days  old  are  used  for  the  first  inoculation, 
and  the  immunization  is  continued  by  using  younger  and  younger  cultures, 
until  finally  a  24-hour  growth,  or  a  dose  of  virulent  blood,  is  inoculated  (Foa 
and  Scabia,  Netter,  Washbourn). 

(iii)  With  stained  cultures. — Sergent  sows  agar  cultures  with  the  blood  of 
a  rabbit  which  has  died  of  a  highly  virulent  pneumococcal  infection.  After 
incubation  the  growth  is  scraped  off  and  made  into  an  emulsion  with  sterile 
normal  saline  solution  containing  a  few  drops  of  a  sterile  aqueous  solution 
of  crystal-violet.  After  about  an  hour  all  the  organisms  are  stained  but  are 
still  living  and  on  sub-culture  give  rise  to  a  new  culture.  A  volume  of  this 
stained  emulsion  equivalent  to  one-tenth  of  an  agar  culture  will  kill  rabbits 
in  12-48  hours  when  inoculated  sub-cutaneously,  but  it  produces  no  effect  if 
inoculated  intra-venously  or  intra-peritoneally.  Rabbits  which  have  received 
several  of  these  harmless  intra-venous  or  intra-peritoneal  inoculations  at 
intervals  of  &-8  days  (the  dose  of  emulsion  inoculated  may  be  progressively 
increased)  are  able  to  resist  the  inoculation  of  a  quantity  of  a  virulent  culture 
which  is  sufficient  to  kill  a  normal  animal  in  less  than  24  hours.  These  rabbits 
are  more  highly  immunized  than  animals  vaccinated  with  sterilized  cultures. 

(iv)  With  bacteriolyzed  cultures.— Neufeld  found  that  if  0- 1-0-2  c.c.  of 
rabbit  bile  was  added  to  2  c.c.  of  a  24-hour-old  broth  culture  of  the 
pneumococcus  the  culture  became  clear  in  15-20  minutes  :  the  cocci  were 
killed  and  bacteriolyzed  and  disappeared  entirely.  The  same  result  is  obtained 
by  using  a  5  per  cent,  solution  of  cholate  or  taurocholate  of  sodium  instead 
of  bile.  Other  micro-organisms,  and  especially  the  pyogenic  streptococci, 
are  not  destroyed  under  these  conditions. 

Neufeld,  Nicolle  and  Adil  Bey,  have  shown  that  rabbits  can  be  highly 
immunized  by  a  single  inoculation  of  2  c.c.  of  a  culture  bacteriolyzed  as 
above. 

5.  Serum  therapy. 

(i)  The  blood  of  animals  naturally  immune  to  the  pneumococcus  has 
neither  therapeutic  nor  immunizing  properties. 

(ii)  The  blood  of  vaccinated  animals  is  not  antitoxic  and  is  incapable  of 
neutralizing  the  toxins  of  the  pneumococcus  either  in  vitro  or  in  vivo  (Issaeff). 

(iii)  The  serum  of  vaccinated  animals  has  no  bactericidal  action  on  the 
pneumococcus  in  vitro.  The  pneumococcus  will  grow  in  the  serum  but 
somewhat  poorly  and  will  retain  its  virulence  though  it  undergoes  some 
morphological  modifications — the  rounded  forms  predominate  and  the 
micro-organisms  are  agglutinated  in  clumps  (Behring  and  Nissen,  Issaeff). 

In  verifying  the  virulence  of  a  pneumococcus  grown  in  the  serum  of  a  vaccinated 
animal  it  is  necessary  to  exclude  a  source  of  error  which  may  arise  from  inoculation 
of  the  entire  culture.  The  culture  is  composed  of  two  parts  :  (1)  the  micro-organisms 
and  (2)  the  serum  which  serves  as  the  culture  medium  and  which,  as  will  be  seen 
immediately,  has  the  power  of  conferring  immunity.  If  the  whole  culture  be 
inoculated  the  animal  being  immunized  by  the  serum  inoculated  with  the  organisms 
will  survive  the  inoculation,  and  the — erroneous — conclusion  will  be  that  the  serum 
is  bactericidal.  One  or  other  of  the  following  devices  must  therefore  be  adopted. 

(a)  Sow  a  trace  of  the  serum  culture  into  a  tube  of  broth  and  inoculate  the  broth 
culture.     This  method  is  open  to  criticism. 

(b)  Pour  the  serum  culture  on  a  sterilized  filter  paper.     Wash  the  organisms  two 
or  three  times  with  sterile  normal  saline  solution.     Collect  the  residue  with  a  brush, 
dilute  it  in  2  c.c.  of  a  normal  saline  solution  and  inoculate  the  emulsion  sub- 
cutaneously. 

(iv)  The  serum  of  vaccinated  animals  has  both  prophylactic  and  thera- 


SERUM  THERAPY  589 

peutic  properties  which  depend  upon  phagocytosis  and  not  upon  any 
bactericidal  or  antitoxic  properties. 

The  serum  of  a  satisfactorily  vaccinated  rabbit  is  very  potent ;  2  or 
4  drops  of  the  serum  of  such  a  rabbit  is  sufficient  to  immunize  a  mouse 
(Foa  and  Carbone)  :  Neufeld  and  Handel's  serum  protects  mice  against 
100,000  fatal  doses  (vide  ante). 

G.  and  F.  Klemperer  arrested  a  pneumococcal  septicaemia  in  a  rabbit 
infected  24  hours  previously  by  inoculating  it  with  8  c.c.  of  the  serum  of  a 
vaccinated  rabbit.  Tizzoni  and  Panichi  prepared  a  serum  which  exhibited 
therapeutic  properties  for  the  rabbit  when  given  intra-venously  in  doses 
of  0'25  per  1000  of  the  weight  of  the  animal. 

The  same  observers  have  shown  that  at  the  moment  of  the  crisis  in  human 
lobar  pneumonia  the  serum  of  the  patient — which  is  toxic  during  the  febrile 
period— acquires  immunizing  and  therapeutic  properties. 

Therapeutic  applications. — Many  observers  relying  upon  the  satisfactory 
results  obtained  in  laboratory  experiments  have  treated  pneumococcal 
infections  in  the  human  subject  with  immunized  rabbit  serum  and  with  the 
serum  of  patients  who  have  survived  the  crisis.  The  results  so  far  obtained 
though  satisfactory  are  not  sufficiently  striking  to.  warrant  the  method  being 
generally  adopted. 

Klemperer  has  shown  that  the  inoculation  of  antipneumococcal  serum  into  the 
healthy  human  subject  is  unaccompanied  by  any  untoward  symptoms.  In  cases 
of  pneumonia  he  inoculated  immunized  rabbit  serum  in  doses  of  6  c.c.  sub-cutaneously 
with  favourable  results.  Foa  and  Carbone  provoke  the  crisis  on  the  fourth  day  by 
giving  two  consecutive  inoculations  of  5  c.c.  of  the  immunized  rabbit  serum.  Foa 
and  Scabia  and  also  Janson  also  noticed  a  rapid  improvement  in  several  cases  of 
pneumonia  following  the  injection  of  5-25  c.c.  of  the  serum  of  a  vaccinated  rabbit. 
Neufeld  and  Handel  recommend  the  inoculation  of  large  doses  of  their  serum : 
Romer  favours  the  use  of  a  polyvalent  serum  :  Pane  uses  immunized  ass  serum. 

Audeoud  inoculated  cases  of  pneumonia  with  2—4  c.c.  of  blood  taken  from  patients 
who  had  passed  the  crisis,  and  in  two  of  them  he  obtained  a  marked  improvement 
and  a  fall  of  temperature  in  15  hours  after  the  inoculation.  Bouchard,  Roger, 
Charrin,  Maraglia'ho  have  obtained  favourable  results  under  similar  circumstances. 

For  the  purposes  of  these  experiments  the  blood  can  be  collected  from  cases  of 
pneumonia  in  the  early  stages  of  convalescence  without  causing  any  inconvenience, 
by  adopting  the  technique  described  on  p.  193. 

6.  Agglutination. 

In  pneumococcal  infections  the  serum  of  man  and  the  lower  animals  acquires 
the  property  of  agglutinating  the  pneumococcus.  The  agglutinating  power 
is  never  other  than  feeble  :  it  cannot  be  demonstrated  by  the  Grunbaum- 
Widal  method  but  only  in  undiluted  serum  cultures  (Bezan9on  and  Griffon). 

The  serum  must  be  collected  aseptically  and  should  not  be  stained  with 
haemoglobin.  It  is  sown  with  a  trace  of  culture  on  normal  rabbit-serum  and 
incubated  at  37°  C.  for  15  or  16  hours. 

The  agglutination  may  be  visible  to  the  naked  eye  or  only  evident  on 
microscopical  examination.  It  is  very  distinct  in  a  drop  of  culture  spread 
out,  dried,  and  stained  (the  "  Medusa  head  "  appearance  of  Bezancon  and 
Griffon).  The  pneumococcus  is  never  agglutinated  by  normal  human,  rabbit 
or  dog  serum. 

In  human  pneumococcal  infections  (pneumonia,  broncho-pneumonia,  sore 
throat,  etc.)  the  agglutination  reaction  is  always  positive,  but  diminishes 
during  convalescence  and  soon  vanishes  altogether. 

Agglutination  is  not  obtained  with  all  strains  of  the  pneumococcus  ;  it  is 
in  a  way  an  individual  and  not  a  specific  property.  Very  often  agglutination 


590  THE   PNEUMOCOCCUS 

is  obtained  with  the  micro-organism  recovered  direct  from  the  infected  person 
while  laboratory  strains  give  negative  results  (Bezangon  and  Griffon). 

7.  Precipitins. 

The  serum  of  persons  suffering  from  pneumonia  and  the  serum  of  immunized 
animals  contain  specific  precipitins  for  filtered  cultures  of  the  pneumococcus. 

8.  Immune  body. 

The  serums  of  pneumonia  patients  (Romer)  and  of  vaccinated  animals 
(Neufeld  and  Handel)  contain  specific  immune  bodies  (Sensibilisatrices) . 

SECTION  IV.— DETECTION,   ISOLATION  AND  IDENTIFICATION 
OF  THE  PNEUMOCOCCUS. 

I.  Man.  A.  During  life. — To  find  the  pneumococcus  in  a  case  of  pneumo- 
coccal  infection  the  following  materials  should  be  examined  : — 

(a)  Sputum. — Collect  the  sputum  with  the  ordinary  precautions  (p.  191) 
and  with  a  stout  wire  remove  a  small  portion  from  the  centre  of  one  of  the 
rusty  nodules. 

1.  Prepare   films   for   microscopic   examination    (vide   infra,    methods   of 
staining). 

2.  It  is  hardly  worth  while  to  sow  cultures  with  sputum  since  the  other 
organisms  present  will  interfere  with  the  growth  of  the  pneumococcus. 

American  observers  have  described  several  ways  of  isolating  the  pneumococcus 
from  sputum  and  saliva.  Burger  recommends  sowing  stroke  cultures  on  glucose - 
serum-agar  (p.  53).  The  agar  is  prepared  with  2  per  cent,  peptone  water  to  which 
2  per  cent,  glucose  is  added,  and  then  made  neutral  to  phenol-phthalei'n.  On  this 
medium  a  characteristic  culture  of  the  pneumococcus  is  obtained  in  18-20  hours — 
i.e.  ring-shaped  colonies  with  raised  edges  and  depressed  centres  :  by  oblique  vision 
the  ring  is  milky,  the  centre  more  transparent. 

3.  Another  portion  is  used  for  inoculation. — Rub  up  a  little  of  the  sputum 
with  sterile  distilled  water  [or  normal  saline  solution]   and  inoculate  the 
emulsion  into  the  root  of  the  tail  of  a  mouse.     If  the  material  be  rich  in 
pneumococci  the  animal  will  die  quickly.     When  the  animal  is  dead  aspirate 
a  little  of  the  heart  blood,  or  take  a  little  of  the  medulla  from  one  of  the  long 
bones — being  careful  to  avoid  contaminating  it — and  sow  tubes  of  agar  and 
broth.     Incubate  at  37°  C. 

Inoculation  for  the  purpose  of  identifying  a  suspected  pneumococcus  ought 
always  to  be  made  into  a  mouse  and  not  into  a  rabbit.  Rabbits  are  not  so 
susceptible  to  the  disease  as  mice,  and  indeed  in  some  cases  of  pneumococcal 
pneumonia  the  sputum  contains  pneumococci  almost  avirulent  for  rabbits 
(Gamaleia). 

(b)  The  inflammatory  exudate. — Collect  the  exudate  by  putting  a  syringe 
needle  into  an  hepatized  area  (p.  198).     The  material  must  be  examined  by 
microscopical  examination  and  by  inoculation.     Bezan9on  and  Griffon  prefer 
to  sow  the  exudate  in  a  young  rabbit's  serum  which  has  not  been  coagulated 
and  to  inoculate  mice  with  the  culture  obtained  after  incubating  for  21  hours. 

(c)  Pus  and  inflammatory  exudates  in  other  situations.— The  technique  is 
the  same  as  for  the  material  from  the  lung. 

(d)  Blood. — The  pneumococcus  is  not  found  constantly  in  the  blood  of 
persons  suffering  from  pneumonia  (Foa,.  Talamon,  Klemperer,  Widal),  though 
it  is  usually  present  in  severe  cases. 

The  examination  in  such  cases  should  preferably  be  made  about  the  fifth  or  sixth 
day.  When  the  disease  is  likely  to  prove  fatal  the  pneumococcus  can  generally 


ISOLATION  OF  THE  PNEUMOCOCCUS  591 

be  found  in  the  blood  during  the  last  days  or  last  hours  of  life.  The  presence  of 
the  organism  in  the  peripheral  circulation  does  not  however  necessarily  imply  a 
fatal  issue. 

The  blood  should  be  examined  microscopically,  culturally  and  by  inoculation 
into  mice.  The  blood  can  be  collected  either  by  pricking  the  finger  or  from 
a  vein  at  the  bend  of  the  elbow  (p.  193).  Widal  recommends  sowing  5  c.c. 
of  blood  in  300-500  c.c.  of  broth  :  by  this  method  he  has  been  able  to  isolate 
the  pneumococcus  from  the  blood  of  persons  suffering  from  pneumonia  in 
one-third  of  the  cases  examined. 

B.  After  death. — (The  post  mortem  examination  should  be  made  as  soon 
as  possible  after  death.)  The  examination  of  the  following  fluids  and  tissues 
will  suffice  for  the  detection  of  the  pneumococcus. 

(a]  The  inflammatory  exudate  in  the  lung.— Cauterize  the  surface  of  an 
hepatized  area.  Introduce  a  Pasteur  pipette  and  aspirate  the  fluid.  With 
the  material  prepare  cover-glass  preparations,  sow  culture  media  and  inoculate 
a  mouse. 

(6)  Sections  of  the  lung. — Put  a  few  small  pieces  of  the  hepatized  area  of 
the  lung  at  once  into  alcohol  or  acid  perchloride.  They  must  be  subsequently 
embedded  in  paraffin,  cut  and  stained  by  the  methods  described  on  pp.  216 
and  219. 

(c)  Pus  and  exudates.- — Collect  the  material  in  the  ordinary  way  and  then 
prepare  films,  sow  cultures  and  inoculate  a  mouse. 

II.  In  animals. — In  animals  the  following  tissues  and  fluids  may  be  examined 
for  the  pneumococcus  viz.  the  blood,  bone  marrow,  pleural  fluid,  peritoneal 
exudate,  pericardial  effusion,  films  and  sections  of  the  internal  organs,  etc. 

Blood  films  and  scrapings  from  tissues  should  be  examined  microscopically. 
Cultures  on  rabbit  serum  sown  with  the  blood,  bone  marrow,  and  exudate 
will  yield  pure  cultures  which  can  be  used  for  inoculation  of  other  animals. 

Portions  of  the  internal  organs  for  sections  should  be  fixed  in  alcohol  or 
acid  perchloride  and  embedded  in  paraffin. 


CHAPTER  XLI. 
STREPTOCOCCI  HOMINIS. 

Introduction. 

Varieties  of  streptococci,  p.  593. 
Section  I. — Experimental  inoculation,  p.  594. 
Section  II. — Morphology,  p.  595. 

1.  Microscopical  appearance  and  staining  reactions,  p.  595.     2.  Cultural  charac- 
teristics, p.  597. 
Section  III. — Biological  properties,  p.  599. 

1.  Vitality  and  virulence,  p.  599.     2.  Bio-chemical  reactions,  p.  600.     Andrewes 
and   Herder's  classification,   p.    601.     3.  Toxins,   p.    602.     Streptocolysin,   p.    603. 
4.  Vaccination,  p.  604.     5.  Serum  therapy,  p.  605.     A.  Monovalent  serums,  p.  606. 
B.  Polyvalent  serums,  p.  608.     6.  Agglutination,  p.  609. 
7.  Bordet-Gengou  reaction,  p.  609. 
Section  IV. — Detection  and  isolation  of  streptococci,  p.  609. 

The  streptococcus  of  Bonome,  p.  610. 

STREPTOCOCCI  were  first  described  by  Pasteur  and  Doleris  who  found  them 
originally  in  the  blood  of  women  suffering  from  puerperal  fever. 

Streptococci  as  the  primary  cause  of  disease. — Streptococci  are  the  primary 
cause  of  many  inflammatory,  suppurative  and  septicsemic  processes.  Thus 
they  are  the  cause  of  puerperal  septicaemia  (Pasteur  and  Doleris)  and  erysipelas 
(Fehleisen)  :  they  are  a  common  cause  of  osteo-myelitis  and  of  purulent 
surgical  affections  and  have  also  been  found  to  be  the  primary  infecting  agent 
in  certain  cases  of  each  of  the  following  among  other  diseases  : — phlebitis, 
broncho-pneumonia,  pleurisy,  peritonitis,  meningitis,  endocarditis,  salpingitis, 
otitis,  and  dermatitis. 

In  all  pathological  conditions  of  the  throat  of  whatever  nature  streptococci 
are  to  be  found  either  as  the  primary  cause  of  the  lesion  or  as  an  associated 
infection. 

[Acute  rheumatism  is  attributed  by  some  observers  to  infection  with  a 
streptococcus  (Beattie  ;  Poynton  and  Paine  and  others).] 

It  is  now  generally  conceded  that  streptococci  are  not  the  specific  cause  of 
scarlet  fever  though  these  organisms  are  almost  constantly  present  as  secon- 
dary infections.  Weaver,  for  instance,  found  streptococci  nearly  always 
present  in  the  throat  during  an  attack  of  scarlet  fever.  Hektoen,  however, 
only  found  streptococci  in  the  blood  in  12  out  of  100  cases  of  scarlet  fever 
which  he  examined. 

Streptococci  as  secondary  infections. — As  secondary  infections  streptococci  are 
not  infrequently  found  complicating  an  already  existing  infective  disease  and  under 
these  conditions  are  even  more  dangerous  than  when  acting  as  primary  infections. 


VARIETIES   OF   STREPTOCOCCI  593 

They  associate  themselves  with  the  primary  cause  of  the  disease  and  increase  its 
virulence,  as  may  happen,  for  instance,  in  influenza,  enteric  fever,  and  diphtheria. 
Streptococci  are  also  found  as  secondary  infections  following  infection  with  the 
pneumococcus,  the  tubercle  bacillus  and  the  bacillus  of  hospital  gangrene.  A  great 
many  of  the  complications  of  scarlet  fever  are  due  to  a  secondary  infection  with 
streptococci. 

Streptococci  occur  in  the  healthy  human  subject  both  on  the  skin  and  in 
those  cavities  which  open  on  the  surface  of  the  body  (alimentary  canal,  nose, 
and,  but  rarely,  in  the  vagina).  They  are  present  also  in  the  saliva,  stools,  etc. 

Streptococci  are  also  the  cause  of  certain  diseases  in  the  lower  animals 
(Chap.  XLII). 

Streptococci  are  also  found  widely  distributed  in  the  air,  soil,  well  water, 
etc.  [and  wherever  found  they  seem  always  derived  directly  or  indirectly 
from  the  animal  body  ( Andre wes)].  The  virulence  of  the  organism  appears 
to  be  rapidly  lost  outside  the  body. 


VARIETIES  OF  STREPTOCOCCI. 

The  large  number  of  pathological  conditions  to  which  the  streptococci  can 
give  origin  and  its  occurrence  as  a  saprophyte  in  the  healthy  human  tissues 
have  for  long  attracted  the  attention  of  bacteriologists.  The  question 
naturally  arises  as  to  whether  or  no  the  streptococci  found  under  these 
different  conditions  are  all  identical. 

For  a  time  there  was  a  tendency  to  regard  the  streptococci  as  including 
several  species  :  Fehleisen,  for  instance,  stoutly  defended  the  specificity  of 
the  streptococci  isolated  from  cases  of  erysipelas  (Streptococcus  erysipelatos) 
and  Rosenbach  stated  that  he  could  with  certainty  distinguish  streptococci 
obtained  from  pus  (Streptococcus  pyogenes)  from  Fehleisen 's  erysipelococcus. 

But  Petruschky  showed  that  the  same  strain  of  streptococcus  would  pro- 
duce indifferently  both  in  man  and  in  the  lower  animals  such  varied  clinical 
conditions  as  erysipelas,  suppuration  and  septicaemia. 

[Clinical  experience  also  rendered  Fehleisen's  and  Rosenbach's  views 
untenable  :  in  clinical  practice  a  streptococcus  from  a  case  of  erysipelas  can 
originate  a  puerperal  infection.  "  One  and  the  same  strain  of  streptococcus 
may  at  different  stages  in  its  career  produce  now  a  rapidly  fatal  septicaemia, 
now  a  spreading  erysipelas,  now  a  localized  suppuration  and  now  no  effect 
at  all  "  (Andrewes  and  Horder).  "  It  may  therefore  be  accepted  as  an 
established  fact  that  a  streptococcal  infection  may  assume  different  clinical 
manifestations  depending  upon  the  resistance  of  the  person  infected  and 
upon  the  structure  of  the  tissue  invaded  and  that  quite  independently  of  the 
source  whence  the  organism  was  originally  derived  "  (Besredka).] 

The  saprophytic  streptococcus  which  is  a  normal  inhabitant  of  the  [mucous 
surfaces  of  the]  human  body  should  not  be  differentiated  from  the  pathogenic 
streptococcus  :  its  saprophytic  character  will  vanish  and  its  virulence  increase 
under  the  influence  of  various  determining  causes  such  as  traumatism,  cold, 
the  ancillary  action  of  other  organisms,  etc.  [Andrewes  however  is  of  a 
contrary  opinion  and  holds  that  the  S.  pyogenes  should  be  clearly  differen- 
tiated from  the  saprophytic  streptococci  (vide  infra).] 

The  marked  pleomorphism  of  the  streptococcus  also  led  many  observers 
to  classify  these  organisms  according  to  their  microscopical  appearances ; 
thus  streptococci  were  divided  into  S.  tenuis,  S.  brevis,  S.  longus,  etc.  But 
microscopical  appearances  and  cultural  characteristics  are  in  the  case  of  the 
streptococci  as  in  the  case  of  the  majority  of  the  bacteria  very  variable  and 
cannot  alone  be  used  as  a  basis  of  classification. 


594  THE  STREPTOCOCCI   OF  MAN 

Chiefly  as  the  results  of  the  experiments  of  Marmorek  and  Aronson  the 
opinion  gained  ground  that  there  was  but  a  single  species  of  streptococcus 
which  according  to  the  conditions  prevailing  might  cause  any  one  of  a  number 
of  different  pathological  conditions. 

[Andre wes  and  Border  from  a  study  of  the  bio-chemical  reactions,  morpho- 
logical appearances  and  other  characteristics  of  a  large  number  of  streptococci 
from  various  sources  have  provisionally  but  without  any  idea  of  finality 
classified  these  organisms  into  six  groups  (vide.  p.  601). 

[Reviewing  these  conflicting  opinions,  it  may  be  said  that  all  observers  are 
at  least  agreed  that  there  are  many  and  important  characteristics  possessed 
by  all  streptococci  in  common,  and  that  the  controversy  hinges  on  the  question 
whether  the  differences  which  undoubtedly  exist  between  various  strains  are 
to  be  regarded  as  of  minor  importance — as  those  who  consider  all  streptococci 
identical  maintain — or  whether  as  the  other  side  insists  these  differences  are 
of  capital  importance  necessitating  a  division  of  the  streptococci  into  species.  ] 

At  the  moment  it  would  seem  best  to  take  up  the  position  that  in  the  case 
of  the  streptococci,  as  in  that  of  the  cholera  vibrio,  there  are  several  races — 
one  might  almost  say  species,  but  an  authoritative  definition  of  the  term  is 
lacking. 

[The  subject  is  not  merely  one  of  academic  interest  but  is  from  the  point 
of  view  of  serum  therapy  of  considerable  practical  importance.] 

It  is  not  within  the  province  of  this  book  to  discuss  the  arguments  brought 
forward  by  the  opposing  theorists — the  subject  is  still  one  of  the  most  acutely 
discussed  problems  in  bacteriology— but  the  differences  between  various 
strains  of  streptococci  will  have  to  be  noticed  as  will  also  the  facts  upon 
which  the  two  sides  base  their  conclusions. 


SECTION  I.— EXPERIMENTAL  INOCULATION. 

Generally  speaking,  streptococci  isolated  from  man,  even  from  severe  infections,  are 
only  slightly  virulent  for  animals.1  Consequently,  the  first  inoculation  of  a  streptococcus 
of  unknown  virulence  should  be  made  with  a  large  dose  of  culture  either  into  the  veins 
or  into  the  peritoneal  cavity.  The  virulence  of  the  organism  for  animals  is  rapidly 
increased  by  passage  through  animals. 

Rabbits.- — Rabbits  are  the  most  useful  animals  for  the  study  of  the  experi- 
mental disease.  For  purposes  of  inoculation  cultures  2  or  3  days  old  should 
be  used  (vide  infra). 

A.  Sub-cutaneous  inoculation. — As  a  rule,  the  animal  is  inoculated  beneath 
the  skin  of  the  ear  ;    this  enables  the  progress  of  the  lesion  to  be  readily 
observed.     According  to  the  virulence  of  the  organism  an  inoculation  of 
10-20  drops  of  culture  will  produce  one  of  the  following  results  : 

(a)  A  small  abscess. 

(b)  A  temporary  erysipelatous  blush. 

(c)  An  erysipelas  involving  the  whole  ear,  possibly  becoming  phlegmonous 
but  not  ending  in  generalization. 

(d)  A  phlegmonous  erysipelas  followed  by  suppurative  arthritis  and  ending 
in  death  in  15-30  days.     Streptococci  cannot,  as  a  rule,  in  this  case  be  found 
either  in  the  articular  pus  or  in  the  blood  after  death. 

(e)  A  rapidly  fatal  septicaemia  ending  in  death  in  a  few  days.     Streptococci 
can  be  found  in  the  blood  post  mortem. 

B.  Intra-venous  inoculation. — Inoculation  of  a  virulent  streptococcus  into 

[x  But  see  later  on  under  the  heading  of  "  Virulence."  Streptococci  from  severe  human 
infections  are  generally  of  the  pyogenes  variety  and  are  pretty  virulent  for  mice  at 
least  (Andrewes).] 


MORPHOLOGY  595 

the  veins  leads  to  a  septicaemia  fatal  in  24-48  hours  ;  streptococci  are  present 
in  the  blood  in  very  large  numbers. 

With  a  less  virulent  organism,  localized  foci  of  suppuration  form  on  serous 
surfaces  ;  from  these  the  animal  may  recover  but  death  often  takes  place 
within  10-20  days.  In  such  cases  streptococci  are  not  found  in  the  blood. 

C.  Intra-peritoneal  inoculation. — Inoculation  into  the  peritoneal  cavity  is 
followed  by  as  severe  an  infection  as  inoculation  into  the  veins.  A  virulent 
streptococcus  will  kill  a  rabbit  in  24-72  hours. 

The  virulence  of  a  given  streptococcus  for  the  rabbit  may  be  indefinitely  increased 
by  passage  through  rabbits.  Marmorek,  for  instance,  was  able  to  increase  the  viru- 
lence of  a  streptococcus  to  such  an  extent  that  one-millionth  and  even  one-thousand- 
millionth  of  a  cubic  centimetre  of  a  broth  culture  was  sufficient  to  kill  a  rabbit 
when  inoculated  into  the  peritoneal  cavity. 

Mice. — Mice  are  almost  as  susceptible  to  infection  with  streptococci  as 
rabbits.  A  virulent  streptococcus  inoculated  beneath  the  skin  will  kill  a 
mouse  in  24—72  hours.  In  the  case  of  less  virulent  organisms  death  does  not 
occur  so  soon  and  is  preceded  by  the  formation  of  abscesses. 

Guinea-pigs. — Guinea-pigs  are  less  susceptible  than  rabbits  and  mice.  The 
inoculation  of  a  virulent  streptococcus  beneath  the  skin  generally  gives  rise 
to  a  local  abscess  which  ultimately  resolves. 

If  the  virulent  passage  streptococcus  of  Marmorek  be  inoculated  into  the  peritoneal 
cavity  in  quantities  of  not  less  than  0'2  c.c.,  a  purulent  peritonitis  and  bacteraemia 
results  which  is  fatal  in  about  15-20  hours. 

Large  animals. — Of  the  larger  animals,  asses  and  horses  are  moderately 
susceptible  to  infection  with  streptococci  [of  average  virulence],  the  latter 
somewhat  less  so  than  the  former.  Dogs  and  sheep,  on  the  other  hand,  are 
relatively  immune. 

Man. — Numerous  experiments  have  been  carried  out  on  the  human  sub- 
ject. The  results  of  inoculation  have  often  been  negative,  but,  on  the  other 
hand,  in  several  instances  a  typical  erysipelas  has  been  produced  and  in  one 
case  the  experiment  terminated  fatally. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance  and  staining1  reactions. 

Streptococci  consist  of  non-motile  cocci  arranged  in  chains. 


*' 


**  %          /    '  :S  r\ 

%%«  "k   '•"'{ 


s,.rv    ; 

•  ••• 

FIG.  279. — Streptococcus.    Film  from 

FIG.  278.— Streptococci  in  pus.    Jenner's  stain.  a  broth  culture      Carbol-crystal- violet. 

(Oc;  2,  obj.  Atfi,  Zeiss.)  (Oc.  III.  obj.  ^th,  Beich.) 


596 


THE  STREPTOCOCCI   OF  MAN 


In  blood  and  pus  the  individual  cocci  measure  O6-1//,  in  diameter,  but  in 
cultures  their  size  is  subject  to  considerable  variation  :  they  occasionally 
assume  a  somewhat  oval  shape. 

The  number  of  cocci  in  the  chains  varies.  Thus,  in  a  typical  streptococcus 
(Streptococcus  erysipelatos  of  Fehleisen,  Streptococcus  pyogene*  of  Rosenbach) 
the  chains,  as  seen  in  preparations  from  blood  and  pus  or  from  a  culture  on 
a  solid  medium,  will  be  found  to  consist  of  six  to  fifteen  cocci  ;  but  when  the 
same  organism  is  grown  on  a  liquid  medium  the  chains  will  show  as  many 
as  fifteen  to  forty  and  more  cocci. 


FIG.  280. — Streptococcal  chains  composed  of 
very  small  cocci. 


FIG.  281. — Diplococcal  streptococci. 


FIG.  282. — Streptococcus  conglomeratus. 


FIG.  283. — Very  long  chains. 


FIGS.  280-283. — Various  forms  of  streptococci. 
(Oc.  2,  obj.  -^th,  Zeiss.) 


Carbol-fuchsin. 


Classification  on  morphological  grounds. — Nothing  is  more  variable  than 
the  number  and  even  the  shape  of  the  cocci  forming  a  streptococcus  chain  : 
and  hence  several  types  of  streptococci  have  been  described  (figs.  280  to  283). 

The  streptococcus  tennis  of  Veillon,  found  in  some  cases  of  sore  throat,  consists 
of  short  chains  of  two  to  six  very  small  oval  cocci.  The  streptococcus  brevis  of  von 
Ldngelsheim,  found  in  the  saliva  in  some  cases  of  pseudo-membranous  inflammation 
of  the  throat  and  also  sometimes  in  pus,  is  a  streptococcus  in  which  the  cocci  are 
arranged  as  diplococci  or  as  chains  of  four  to  six  diplococci.  [The  streptococcus 
longus  (von  Lingelsheim)  consists  of  long  chains,  and  includes  most  of  the  strains 
virulent  for  man.]  The  streptococcus  conglomeratus,  which  Kurth  considered  to 


CULTURAL  CHARACTERISTICS 


597 


B 


FIG.  284. — Different  appearances 
presented  by  the  same  streptococcus 
(A)  when  grown  on  agar  and  (T 


be  the  specific  cause  of  scarlet  fever,  is  characterized  by  its  tendency  to  form  long 
chains  massed  together  like  staphylococci. 

Many  authors  regard  these  differences  in  form  as  accidental.  Marmorek  who,  in 
common  with  many  other  observers,  holds  that  all  streptococci  are  identical,  has 
shown  that  the  number,  shape  and  arrangement  of 
the  cocci  in  the  chains  can  be  altered  at  will  by 
modifying  the  composition  of  the  culture  medium 
(fig.  284). 

Practically  all  bacteriologists  are  now  agreed 
that  these  purely  morphological  differences  are 
insufficient  to  justify  a  differentiation  into 
species. 

Staining  reactions. — Streptococci  stain  readily 
with  the  ordinary  basic  aniline  dyes  and  are 
gram -positive. 

It  is  to  be  noted  however  that  there  are  streptococci 
which  do  not  stain  by  Gram's  method.  Von  Lingel- 
sheim,  for  instance,  described  a  gram-negative 
streptococcus,  Etienne  found  a  similar  strain  in  a 

«  ,  -,  T  .  -,  .  P  \-r*-J  wiicjj.  givs  vr  11  \ju.  agai   an\i  t  jj  i  wucj 

case  of  sore  throat,  and  other  instances  of  gram-    grown  in  serum-broth.  (AfterZenoni.) 
negative  streptococci  have  been  recorded.     Lemoine 

isolated  a  gram-variable  streptococcus  from  a  case  of  erysipelas.  [But  Andrewes 
and  Horder,  and  Gordon,  found  no  gram-negative  streptococci  during  their  researches 
provided  they  used  decently  vigorous  cultures.  The  reaction  to  Gram's  stain 
depends  partly  upon  whether  Gram's  method  or  the  individual  observer's  modi- 
fication be  employed  and  partly  upon 
the  age  of  the  culture.  It  is  far  from 
uncommon  to  find  dead  and  dying  cocci 
in  a  chain  gram-negative  (Andrewes).] 

2.  Cultural  characteristics. 

Conditions  of  growth. — Streptococci 
are  facultative  aerobic  organisms  [and 
grow  anaerobically  quite  as  well  as 
aerobically  (Andrewes).]  Growth 
takes  place  between  20°  and  46°  C., 
the  optimum  temperature  being  37°- 
38°  C.  [Andrewes  and  Horder  point 
out  that  all  pathogenic  streptococci 
grow  best  at  the  temperature  of  the 
body  but  while  some  are  capable  of 
vigorous  growth  at  20°  C.  others  are 
altogether  incapable  of  growth  at  that 
temperature.] 

Streptococci  require  a  neutral  or 
slightly  alkaline  medium  and  grow 
better  on  media  containing  serum  or 
blood  than  on  the  ordinary  laboratory 
media. 

Characters  of  growth.  Broth. — A 
typical  pathogenic  streptococcus  does 
not  render  broth  turbid.  After  incubating  for  24  hours  at  37°  C.  the  growth 
takes  the  form  of  a  light  flocculent  precipitate  adhering  to  the  walls  of  the 
tube  which,  increasing  in  amount  for  the  next  3  or  4  days,  ultimately  falls 
to  the  bottom  forming  a  somewhat  abundant  greyish  deposit :  the  reaction 
of  the  medium  is  then  distinctly  acid  (lactic  acid). 


FIG.  285.  —  Strepto- 
coccus pyogenes.  Stab 
culture  in  gelatin  (5  days 
at  22°  C.). 


FIG.  286.  —  Strepto- 
coccus pyogenes.  Surface 
culture  on  agar  (4  days 
at  37°  C.). 


598  THE   STREPTOCOCCI   OF  MAN 

Some  streptococci,  however,  and  especially  the  short  chain  forms  produce  a  turbid 
growth  after  incubating  for  24  hours  at  37°  C.  :  later  finely  granular  masses  appear 
in  the  medium  and  these  after  3-5  days'  growth  fall  to  the  bottom  leaving  the 
medium  clear. 

[Aronson  uses  glucose-broth  for  growing  streptococci]  :  and  according  to 
Ucke,  5  per  cent,  glycerin-broth  is  a  very  useful  medium  for  the  growth  of 
the  organism. 

Marmorek's  phenomenon. — If  a  broth  culture  of  a  streptococcus  be  filtered 
and  the  filtrate  be  then  resown  either  with  the  same  or  with  another  strain 
of  streptococcus  the  organism  fails  to  grow  while  any  other  organism,  e.g.  a 
staphylococcus,  a  pneumococcus,  etc.,  will  grow  abundantly.  This  failure  to 
grow  in  a  filtered  broth  culture  is  common  to  all  streptococci  found  in  human 
disease  processes  ;  it  is  to  be  noted,  however,  that  streptococci  isolated  from 
cases  of  scarlet  fever  may  give  a  very  poor  growth.  The  streptococcus  of 
strangles  (Chap.  XLII.)  can,  by  means  of  this  test,  be  readily  distinguished 
from  all  other  streptococci :  in  a  streptococcal  filtrate  this  organism  grows 
almost  as  well  as  any  other  organism.  Marmorek  quotes  this  experiment 
as  a  valuable  argument  in  favour  of  his  view  that  all  streptococci  of  human 
origin  are  identical. 

Liquid  seriim. — Fresh  rabbit  serum  is  a  very  favourable  culture  medium, 
but  ascitic  fluid  and  bovine  and  horse  serum  alone,  without  any  addition, 
are  not  particularly  useful ;  the  growth  on  the  latter  media  is  poorer  than  in 
broth  but  otherwise  has  similar  characteristics. 

Serum-broth  and  blood-broth. — Marmorek  recommends  the  following  media 
as  giving  rich  and  virulent  cultures. 

(a)  Equal  parts  of  peptone-broth  and  human  serum.     The  best. 

(/?)  Equal  parts  of  peptone-broth  and  ascitic  or  pleural  fluid. 

(y)  One  part  of  peptone-broth  and  two  parts  of  horse,  mule,  or  donkey 
serum. 

The  characteristics  of  the  growth  are  similar  to  those  on  broth. 

[Besredka  keeps  his  stock  cultures  of  streptococci  in  a  mixture  consisting  of 
equal  parts  of  Martin's  broth  and  heated  (56°  C.  for  ^  hour)  horse  serum.  This 
observer  finds  that  the  organisms  remain  alive  hi  this  medium  for  a  long  time  and 
retain  their  virulence  well.] 

Milk. — When  streptococci  are  grown  in  milk  the  medium  is  coagulated 
usually  after  4  or  5  days,  occasionally  later.  In  some  cases  no  clot  is  formed. 
[Andrewes  and  Horder  found  that  among  the  streptococci  pathogenic  for  man 
studied  by  them,  the  commonest,  which  they  describe  as  S.  pyogenes,  does 
not  coagulate  milk.  On  the  other  hand  those  which  they  refer  to  the  groups 
S.  salivarius,  S.  anginosus,  S.  fcecalis  all  produce  clot  in  milk  (vide  infra]. 
Schottmiiller's  Streptococcus  pyogenes  does  not  clot  milk.  ] 

Gelatin. — The  growth  in  gelatin  stab  culture  is  poor  and  consists  of  isolated, 
circular,  opaque-white  colonies  barely  the  size  of  a  pin's  head.  Growth 
ceases  about  the  fifth  day  and  the  organisms  soon  die.  [As  already  pointed 
out  some  streptococci  do  not  grow  at  all  at  the  temperature  of  solid  gelatin, 
20°  C.] 

[As  a  rule]  the  medium  is  not  liquefied.  [Andrewes  and  Horder,  however, 
isolated  a  few  pathogenic  streptococci  which  liquefied  gelatin.  S.  fcecalis 
(vide  infra]  liquefies  gelatin  far  more  commonly  than  S.  pyogenes.] 

Agar. — After  incubating  for  12-24  hours  at  37°  C.  a  number  of  small  whitish 
colonies,  resembling  grains  of  semolina,  appear  along  the  line  of  sowing.  On 
further  incubation  the  colonies  increase  in  size,  and  may  become  confluent 
forming  a  somewhat  thin,  semi-opaque,  greyish  band  with  more  or  less  sharply 
defined  edges.  The  culture  soon  dies  out. 


BIOLOGICAL  PROPERTIES  599 

[Blood-agar. — Schottmiiller  used  a  medium  consisting  of  2  parts  of  sterile 
human  blood  mixed  with  5  parts  of  ordinary  agar. 

[By  the  characteristics  of  the  growth  on  this  medium,  Schottmiiller  considered 
that  streptococci  could  be  divided  into  three  groups. 

[I.  Streptococcus  pyogenes  vel  erysipelatos  which  is  a  long-chained  form  giving 
rise  to  greyish  colonies  and  hsemolyzing  the  blood. 

[2.  Streptococcus  mitior  vel  viridans  growing  in  short  chains  and  giving  origin  to 
greenish  colonies  but  producing  very  slight  haemolysis. 

[3.  Streptococcus  mucosus  consisting  of  capsulated  organisms  and  giving  rise  to 
colonies  of  a  mucous,  slimy  consistency.  ] 

Coagulated  serum. — The  colonies  are  more  often  discrete  otherwise  the 
growth  is  the  same  as  on  agar. 

Marmorek  recommends  agar  over  which  a  drop  or  two  of  human  serum  has 
been  run. 

[Serum-agar. — To  obtain  large  quantities  of  growth  for  immunizing  horses,  Bes- 
redka  uses  Roux's  bottles  22  x  11  cm.  and  sows  on  agar  which  is  watered  1  hour 
before  sowing  with  1-1 -5  c.c.  of  heated  horse  serum  (56°  C.  for  |  hour).] 

Potato. — No  growth  apparent  to  the  naked  eye  takes  place  on  potato  though 
if  the  surface  of  the  medium  be  scraped  and  the  scrapings  examined  under  the 
microscope  it  is  evident  that  some  multiplication  has  taken  place.  The 
chains  are  always  short.  Marot  has  described  a  streptococcus  giving  rise 
on  this  medium  to  small  discrete  whitish  colonies  visible  to  the  naked  eye. 

SECTION  HI.— BIOLOGICAL  PROPERTIES. 
1.  Vitality.    Virulence. 

Vitality. — Under  aerobic  conditions,  streptococci  soon  die  out  in  artificial 
culture.  Cultures  more  than  a  fortnight  old  often  fail  when  resown. 

When  sub-cultivated  from  agar  to  agar  the  organism  soon  dies  out,  but  in 
broth  sub-cultures  can  be  maintained  for  a  much  longer  time.  Marmorek's 
[and  Besredka's]  serum  media  are  better  for  keeping  the  organism  alive. 
Sub-cultures  from  agar  can  often  be  obtained  by  sowing  in  serum-broth  when 
they  fail  on  ordinary  media.  Meyer  and  Ruppel  kept  streptococci  in  human 
blood  sealed  up  in  tubes  in  the  ice  chest  and  found  that  they  retained  both 
their  vitality  and  virulence  for  years. 

In  culture,  streptococci  are  readily  destroyed.  A  temperature  of  52°  C. 
for  1  hour  or  100°  C.  for  an  instant  suffices  to  kill  the  organisms.  Antiseptics, 
even  the  weakest,  are  powerfully  bactericidal ;  chloroform  vapour,  for 
example,  sterilizes  cultures  of  streptococci  almost  at  once. 

In  pus,  especially  when  dried,  the  organism  is  more  resistant.  A  tem- 
perature of  100°  C.  for  a  few  minutes  kills  it  but  it  will  resist  ordinary  anti- 
septics for  a  fairly  long  time. 

[Some  of  the  non-pathogenic  forms  of  streptococci — the  S.  equinus  of  Andrewes  and 
Horder  (vide  infra) — are  highly  resistant  and  will  withstand  drying  on  garnets  for  several 
months.  ] 

Virulence.— Streptococci  soon  lose  their  virulence  when  grown  on  the 
ordinary  culture  media,  but  the  virulence  may  be  maintained  by  keeping  the 
organism  under  anaerobic  conditions  or  in  broth  containing  calcium  carbonate 
or,  more  certainly  still,  in  human  blood  or  in  the  serum  media  of  Marmorek 
[and  Besredka].  The  virulence  is  not  raised  by  sub-culturing  in  the  latter 
media  but  is  maintained  for  several  generations. 

Streptococci  isolated  from  human  lesions  (suppurative  lesions,  sore  throats, 
septicaemias,  etc.)  are,  as  .a  rule,  very  slightly  virulent  for  the  lower  animals 
and  may  even  be  totally  non- virulent  for  rabbits  and  mice  :  Vei lion's  Strepto- 
coccus tennis  and  von  Lingelsheim's  Streptococcus  brevis  are  examples  of  the 


600  THE   STREPTOCOCCI   OF  MAN 

latter  type.  No  relation  has  been  established  between  the  virulence  for  man 
and  the  virulence  for  the  lower  animals. 

[Andrewes  and  Horder  found  that  streptococci  of  their  pyogenes  and 
anginosus  groups,  which  include  145  out  of  194  strains  examined,  were  patho- 
genic to  mice  when  first  isolated  from  the  body  ;  those  of  the  salivarius  and 
JoBcalis  groups  were  non-pathogenic.  In  their  experience  a  streptococcus 
may  be  the  cause  of  a  septicaemia  or  of  an  inflammatory  affection  in  man 
and  yet  produce  little  or  no  effect  when  inoculated  into  a  rodent,  and  they 
formed  the  opinion  that  while  there  was  a  general  correspondence  in  patho- 
genic effect  upon  man  and  rodents  it  was  not  universal.] 

Method  of  increasing  the  severity  of  experimental  streptococcal  infections. — 
(a)  The  pathogenic  power  of  a  streptococcus  for  a  rabbit  may  be  increased  by 
inoculating  with  it  sterilized  culture  of  Proteus  vulgaris  (Roger  and  others). 

(b)  Vincent  has  shown  that  the  streptococcus  is  more  virulent  for  laboratory 
animals  when  associated  with  the  typhoid  bacillus  and  its  products. 

A  streptococcal  culture,  0'25  c.c.  of  which  inoculated  into  the  veins  of  a  rabbit 
caused  no  rise  of  temperature,  led  to  a  fatal  septicaemia  in  an  animal  which  had, 
previously  to  inoculation  with  the  streptococcus,  been  inoculated  with  a  culture 
of  the  typhoid  bacillus.  Cultures  of  the  typhoid  bacillus,  sterilized  by  filtration, 
and  inoculated  at  the  same  time  as  a  streptococcus  render  the  latter  much  more 
virulent.  A  highly  immune  animal  such  as  the  guinea-pig  dies  of  a  streptococcal 
septicaemia*  if  it  be  inoculated  intra-peritoneally  with  a  mixture  consisting  of  2  c.c. 
of  a  filtered  culture  of  the  typhoid  bacillus  and  1  c.c.  of  a  streptococcus  the  virulence 
of  which  has  not  been  artificially  raised. 

Exaltation  of  virulence. — (a)  The  method  recommended  for  obtaining  a  very 
virulent  virus  for  the  preparation  of  toxin  is  that  of  Marmorek  who  increases 
the  virulence  of  the  organism  by  passage  through  rabbits.  The  technique 
is  as  follows  : 

Inoculate  a  fatal  dose  of  the  streptococcus  into  the  ear  vein  of  a  rabbit.  As  soon 
as  the  animal  is  dead,  sow  some  of  the  heart  blood  in  a  tube  of  human-blood-serum. 
Incubate  at  37°  C.  for  48  hours  and  then  inoculate  a  second  rabbit  with  the  culture. 
Sow  a  second  culture  and  inoculate  a  third  rabbit  and  so  on  indefinitely.  The  viru- 
lence can  only  be  maintained  by  repeated  passage  through  animals.  After  passage 
experiments  extending  over  2  months  Marmorek  found  that  the  streptococcus  was 
so  virulent  that  0-000,000,000,01  c.c.  sufficed  to  kill  a  rabbit.  (This  exceedingly 
minute  dose  diluted  with  1  c.c.  of  broth  and  inoculated  into  the  peritoneal  cavity 
led  to  death  from  septicaemia  in  30  hours.) 

(b)  In  a  similar  manner,  Aronson  increased  the  virulence  of  streptococci 
by  passage  through  mice. 

2.  Bio-chemical  reactions. 

Formation  of  indol. — Streptococci  do  not  form  indol  in  culture. 

[Neutral  red. — Using  ordinary  broth  tinted  with  the  dye  it  has  been  found 
that  some  streptococci  after  anaerobic  cultivation  for  48  hours  change  the 
colour  of  the  medium  from  cherry  red  to  orange  green,  while  others  leave  the 
original  colour  unaltered  (vide  infra). 

[Fermentation  reactions. — Gordon  tested  ten  "  representative  "  streptococci 
obtained  from  disease  processes  and  other  sources  upon  33  substances  of  the 
carbohydrate,  glucoside  and  polyatomic  alcohol  series. 

The  medium  was  made  up  thus  : 

Lemco,        ...  1  per  cent. 

Peptone,     -          -  1 

Test  organic  substance,  1 

Sodium  bicarbonate,     ......  O'l 

10  per  cent,  watery  solution  of  ordinary  solid  litmus,        -  10 

Water, 87 

and  the  cultures  were  incubated  for  3  days  at  37°  C. 


BIO-CHEMICAL   REACTIONS  601 

[In  glucose,  fructose,  mannose,  galactose,  maltose  and  dextrin  all  the  ten 
streptococci  tested  gave  an  acid  reaction. 

[No  acid  was  formed  by  any  of  the  streptococci  in  starch,  glycogen,  arabin, 
convolvulin,  hesperidin,  jalapin,  methyl  glucoside,  saponin,  glycol,  erythrite 
or  dulcite. 

[In  the  presence  of  the  following  substances  some  gave  an  acid  reaction. 
Rhamnose  (iso-dulcite),  saccharose,  lactose,  raffinose,  inulin,  amygdalin, 
arbutin,  coniferin,  digitalin,  helicin,  populin,  salicin,  syringin,  glycerin,  sorbite, 
and  mannite. 

[With  a  view  to  classifying  streptococci  according  to  their  bio-chemical 
characters,  Gordon  selected  from  among  these  last-named  substances  the 
following  :  saccharose,  lactose,  raffinose,  inulin,  salicin,  coniferin  and  mannite. 
He  took  into  account  also  the  reactions  in  neutral  red  broth  and  litmus  milk. 
The  reactions  with  these  nine  media  constitute  Gordon's  "metabolic"  tests 
for  the  streptococci. 

[Over  1200  strains  of  streptococci  have  now  been  submitted  to  Gordon's 
tests.  300  from  normal  saliva  (Gordon),  300  from  normal  human  stools 
(Houston),  200  from  air  (Gordon),  172  from  milk  (Houston),  over  200  from 
disease  processes  (Andrewes  and  Horder)  as  well  as  a  number  from  air, 
sewage,  milk  and  the  intestines  of  carnivorous  and  herbivorous  animals 
(Andrewes). 

[Andrewes  and  Horder  have  summarized  the  results  obtained  and  conclude 
that  while  in  themselves  the  chemical  tests  are  too  arbitrary  to  form  a 
basis  for  a  systematic  classification  yet  taken  in  conjunction  with  other 
characters  "  they  afford  a  clue  to  the  nature  of  any  given  streptococcus 
which  is  invaluable."  And  as  a  result  of  their  investigations  these 
observers  consider  it  possible  to  roughly  classify  all  streptococci  into  seven 
groups : 

[A.  Streptococcus  equinus. — A  saprophytic  group  apparently  derived  from  the 
herbivorous  intestine.  It  is  chiefly  found  in  air,  dust  and  horse  dung :  on  rare 
occasions  also  in  human  saliva,  human  stools  and  urine.  So  far  as  is  known  it  is 
totally  devoid  of  pathogenic  properties  for  man. 

[B.  Streptococcus  mitis. — Essentially  saprophytic  and  occurring  chiefly  in  human 
saliva  and  faeces  but  occasionally  associated  with  disease.  It  is  non-pathogenic  and 
is  never  associated  with  suppuration. 

[C.  Streptococcus  pyogenes. — This  group  has  as  its  type  the  classical  organism 
first  described  by  Fehleisen  as  streptococcus  erysipelatos.  It  is  pathogenic  for  man 
and  is  typically  associated  with  suppurative  processes.  It  is  only  occasionally 
found  as  a  saprophyte. 

[D.  Streptococcus  salivarius. — Characteristically  found  in  saliva  though  it  is 
common  in  the  intestine.  This  is  the  type  which  many  authors  describe  as  the 
streptococcus  brevis  of  the  mouth.  In  many  ways  it  is  related  to  the  pneumococcus 
but  is  certainly  not  identical  with  it  being  usually  non-pathogenic.  According  to 
Rosenau  S.  salivarius  is  a  modified  pneumococcus. 

[E.  Streptococcus  anginosus. — A  pathogenic  form  of  S.  salivarius  "  and  seems  to 
have  a  special  connexion  with  inflammation  of  the  fauces  and  with  scarlet  fever." 
It  occurs  in  other  forms  of  sore- throat  and  in  the  alimentary  canal,  and  also  but 
less  commonly  in  other  diseased  conditions. 

[F.  Streptococcus  faecalis. — This  is  the  typical  intestinal  streptococcus  of  man 
and,  according  to  Gordon,  is  not  found  in  normal  saliva.  It  is  sometimes  found  in 
disease  processes  notably  in  cystitis. 

[G.  The  Pneumococci. — The  distinguishing  feature  of  this  group  is  the  presence 
of  a  capsule  when  growing  in  the  animal  body  and  on  certain  special  media.  The 
cocci  frequently  form  chains  and  for  this  reason,  and  because  the  capsule  is  not 
formed  on  ordinary  media,  Andrewes  and  Horder  are  of  opinion  that  "  there 
seems  no  justification  for  removing  the  pneumococci  from  the  genus  strepto- 


602 


THE   STREPTOCOCCI   OF   MAN 


[The  chemical  characters,  pathogenic  properties,  etc.  of  each  of  the  groups 
are  shown  in  the  subjoined  table  which  has  been  compiled  from  Andre wes 
and  Horder's  papers  in  the  Lancet,  1906  ii. 


GORDON'S  "METABOLIC"  TESTS. 

d 

IN  MAN. 

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d 

li 

i 

51 

1 

.  .2 

,38 

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TYPES. 

1 

O  '~~- 

1 

s 

i 

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5 

II 

1| 

*J 

|§ 

•S 

—   *"* 

9 

O 

0 

Q 

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=2 

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O 

"§ 

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"o 

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a 

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'S 

S 

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«  * 

3 

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1 

a 

3 

a 

1 

8 

1 

o 

ss 

.„! 

S.  equinus    - 

• 

. 

+ 

• 

•- 

• 

+ 

+ 

• 

• 

Medius 

Non- 
pathogenic. 

Almost 

S.  mitis 

. 

± 

+ 

+ 

. 

± 

± 

. 

+ 

Brevis 

never 

pathogenic. 

S.  pyogenes 

• 

• 

+ 

+ 

' 

• 

* 

• 

4- 

Longus 

71 

Cases 

20 

Cases 

+ 

S.  salivarius 

+ 

± 

+ 

4- 

± 

• 

• 

. 

• 

± 

Brevis 

26« 
Cases 

7(1) 
Cases 

• 

S.  anginosus 

+ 

i 

* 

+ 

* 

Longus 

16 

Cases 

38« 
Cases 

* 

13 

3 

S.  fsecalis 

+ 

+ 

+ 

+ 

• 

• 

+ 

+ 

+ 

+ 

Brevis 

Cases 

Cases 

• 

Q4. 

Pneumococci 

± 

• 

+ 

+ 

+ 

± 

• 

• 

• 

• 

Brevis 

o*± 

Cases 

+ 

[It  is  not  pretended  by  the  authors  that  this  is  a  hard  and  fast  classification, 
on  the  contrary,  it  is  frankly  admitted  that  one  group  fades  insensibly  into 
another  :  "  we  venture  to  believe  that  some  such  conception  of  the  streptococci 
as  we  have  set  forth  is  preferable  to  the  idea  that  they  are  all  one  kind  or  that 
they  present  a  hopeless  chaos  "  (Andrewes  and  Horder). 

3.  Toxins. 

1.  Roger  grew  a  streptococcus  anaerobically  in  meat  broth  at  30°  C.  for  5 
days  and  then  filtered  the  culture  through  porcelain. 

When  inoculated  into  the  veins  of  a  rabbit  in  doses  of  15-20  c.c.  per  kg.  of  body 
weight  the  filtrate  caused  diarrhoea  and  wasting  and  the  animal  died  in  2  days. 
Rabbits  inoculated  with  5-12  c.c.  of  the  filtrate  and  subsequently  (15-30  days  later) 
with  a  virulent  culture  died  more  quickly  than  control  animals.  On  the  other  hand, 
if  the  filtrate  were  heated  to  104°  C.  and  then  inoculated  into  the  veins  in  doses 
of  5-30  c.c.  it  produced  a  certain  degree  of  immunity.  From  this  it  must  be  assumed 
that  the  filtrate  contained  two  substances,  one,  thermolabile  at  104°  C.  and  precipitable 
by  alcohol,  which  was  toxic  and  predisposed  to  infection,  the  other,  thermostable  at 
104°  C.  and  possessing  immunizing  properties. 

1  Mostly  terminal  or  chronic  infections. 

2  Principally  from  cases  of  scarlet  fever. 


STREPTOCOLYSIN  603 

2.  Marmorek  sowed  a  passage  organism  of  increased  virulence  on  human  - 
serum-broth  and  after  incubating  for  3  months  filtered  it  through  porcelain. 

The  filtrate  inoculated  in  doses  of  1  c.c.  into  a  rabbit  weighing  2  kg.  killed  the 
animal  in  3  or  4  days.  The  virulence  of  the  toxin  was  diminished  by  heating  at 
58°  C. 

Marmorek  prepared  another,  more  powerful,  toxin  by  growing  the  organism 
in  broth  to  which  a  little  leucin  and  glycocoll  and  some  guinea-pig  leucocytes 
had  been  added. 

3.  Bonome  and  Bombicci  are  of  opinion  that  the  toxin  of  the  streptococcus 
is  an  endotoxin.1     By  treating  streptococci  with  10  volumes  of  a  0'75  per  cent, 
solution  of  potash,  filtering  and  precipitating  with  a  1  per  cent,  aqueous 
solution  of  acetic  acid,  they  obtained  a  product  which  was  toxic  for  rabbits. 
Rabbits  inoculated  with  gradually  increasing  doses  of  the  filtrate  acquired  a 
slight  degree  of  immunity  against  the  organism  used  in  the  preparation  of 
the  toxin  but  not  against  any  other  strain. 

Hsemolysin.    (Streptocolysin.) 

Marmorek  observed  that  the  streptococcus  is  capable  of  producing  an 
haemolysin  in  the  tissues  of  living  animals  and  in  this  way  appeared  to  differ 
from  all  other  organisms.  He  considered  that  all  streptococci  of  human 
origin  were  haemolytic,  and  adduced  this  opinion  in  further  support  of  his 
reasons  for  regarding  all  such  streptococci  as  identical. 

[Schlesinger  however  has  described  streptococci,  both  pathogenic  and  non- 
pathogenic,  which  were  not  haemolytic.]  Besredka  also  has  isolated  strains 
of  streptococci  which  not  only  did  not  produce  haemolysis  in  vitro  but,  and 
this  is  very  uncommon,  were  non-haemolytic  in  vivo.  [Schottmiiller  observed 
the  same  fact  and,  as  has  already  been  pointed  out,  divides  streptococci  into 
three  groups  according  to  the  extent  of  haemolysis  produced  when  grown  on 
blood-agar  (p.  599).  Andrewes  and  Horder  find  that  among  the  pathogenic 
streptococci  examined  by  them  those  referable  to  their  "  pyogenes  "  and 
"  anginosus  "  groups  alone  produce  haemolysis  on  blood-agar,] 

Besredka  has  investigated  the  nature  of  the  streptococcal  haemolysin. 

Cultures  in  Marmorek's  ascitic  broth  are  powerfully  haemolytic  but  lose 
this  property  on  being  filtered  through  a  Chamberland  bougie.  [Besredka, 
however,  points  out  that  the  phenomenon  of  haemolysis  in  unfiltered  cultures 
is  of  only  minor  importance  ;  most  micro-organisms  are  more  or  less  haemolytic 
in  artificial  culture  but,  curiously  enough,  lose  this  property  on  filtration.  In 
the  case  of  a  few  streptococci,  however,  a  specific  haemolysin  ( Streptocolysin) 
is  formed  which  is  filtrable,  and  for  the  test  of  haemolysis  to  be  of  value  filtered 
cultures  should  be  used.] 

Preparation  of  Streptocolysin. — Besredka  recommends  the  following  technique  for 
the  preparation  of  a  powerful  Streptocolysin  : 

Inject  a  few  drops  of  a  24-hour  culture  of  a  streptococcus  in  ascitic-broth  beneath 
the  skin  of  a  rabbit.  Next  day,  as  soon  as  the  animal  is  dead,  ascertain  that  the 
red  cells  are  dissolved,  and  then  sow  two  or  three  drops  of  the  heart's  blood  into 
a  tube  of  pure  rabbit-serum  or  equal  parts  of  broth  and  horse-serum.  Incubate 
for  24  hours,  then  add  an  equal  volume  of  normal  saline  solution  and  filter  through 
a  Chamberland  bougie. 

The  filtrate  readily  dissolves  rabbit,  human,  guinea-pig  and  sheep  red-cells 
and  to  a  less  extent  those  of  the  horse  and  bovine  animal :  it  is  most  actively 
haemolytic  at  37°  C.  It  is  not  toxic.  Attempts  to  immunize  animals  with 
the  object  of  preparing  an  anti-haemolysin  have  failed. 

At  the  temperature  of  the  laboratory,  Streptocolysin  deteriorates  in  a  few 

1  Simon  considers  that  there  is  both  an  endotoxin  and  an  extra-cellular  toxin. 


604  THE  STREPTOCOCCI   OF  MAN 

days  and  is  destroyed  in  about  a  fortnight  at  15°-18°  C.  A  temperature  of 
55°  C.  for  30  minutes  has  no  effect,  but  exposure  to  this  temperature  for 
10  hours  or  to  70°  C.  for  2  hours  totally  destroys  its  properties. 

4.  Vaccination. 

(a)  With  toxin. — Attempts  to  immunize  animals  by  inoculating  them  with 
heated  cultures  (even  when  cultures  heated  to  60°  C.  were  used)  have  given 
no  conclusive  results  (Marmorek,  Schonke witch). 

Marmorek  tried  to  immunize  a  horse  by  inoculating  it  with  increasing  doses 
of  a  toxin  which  he  had  prepared  (vide  ante)  and  which  killed  rabbits  in  doses 
of  1  c.c.  A  horse  weighing  300  kg.  was  inoculated  with  1260  grams  of  the 
toxin  in  14  injections  carried  out  over  a  period  of  2  months.  There  was  only 
a  slight  reaction  and  the  serum  proved  to  be  of  very  little  value. 

(ft)  With  living  cultures. — This  is  the  most  certain  and  rapid  method  of 
immunization. 

Rabbits. — Marmorek  immunized  rabbits  by  inoculating  them  first  with  old 
cultures  then  with  increasing  doses  of  virulent  cultures. 

The  most  satisfactory  results  were  secured  in  those  cases  in  which  the  animals 
were  in  the  first  instance  inoculated  beneath  the  skin  of  the  ear  with  an  organism 
sufficiently  virulent  to  cause  a  severe  erysipelas  :  some  of  the  animals  succumbed 
after  the  first  inoculation.  But  even  those  animals  which  were  satisfactorily  vac- 
cinated by  this  method  were  not  immune  to  the  inoculation  of  the  highly  virulent 
passage  streptococci  of  which  the  lethal  dose  was  0-000,000,01  c.c.  The  serum 
of  these  rabbits  while  immunizing  other  rabbits  against  infection  with  the  strepto- 
coccus which  had  been  used  for  immunization,  exhibited  as  might  have  been  antici- 
pated no  prophylactic  properties  against  a  passage  streptococcus. 

MironofE  had  similar  results  with  rabbits  treated  first  with  sterilized  cultures 
then  with  increasing  doses  of  virulent  cultures. 

Grromakowski  inoculated  rabbits  in  the  peritoneal  cavity  first  with  an  old 
culture  heated  to  100°  C.  then  with  an  old  culture  unheated  (5-10  c.c.)  and 
lastly  with  increasing  doses  (1-10  c.c.)  of  virulent  cultures.  The  animals 
had  fifteen  inoculations,  a  fortnight  intervening  between  each  inoculation. 
In  the  end  the  rabbits  were  immune  against  an  intra-peritoneal  inoculation 
of  30  c.c.  of  a  virulent  culture. 

Large  animals. — Marmorek  immunized  an  ass,  an  horse  and  a  sheep  by 
inoculating  small  doses  of  an  extremely  virulent  streptococcus  beneath  the 
skin  :  as  soon  as  the  animal  recovered  it  was  re-inoculated  with  a  larger 
dose.  Each  inoculation  was  followed  by  a  marked  reaction. 

Horses. — Begin  with  an  inoculation  of  0*75-2  c.c.  of  a  serum-broth  culture  of  a 
virulent  organism  beneath  the  skin  of  the  neck.  (When  large  quantities  of  culture 
are  required  ascitic  fluid  or  ass  serum  may  be  used  instead  of  human  blood- serum.) 
The  animal  reacts  violently ;  the  temperature  reaches  40°  C.  and  this  rise  is  accom- 
panied by  the  development  of  a  firm  oedema  at  the  site  of  inoculation.  To  obtain 
an  efficient  serum  it  is  essential  that  the  animals  should  be  made  to  react  violently. 
When  the  animal  has  recovered  completely  re-inoculate  it  with  a  double  dose  (5  c.c. 
for  example)  of  a  virulent  culture.  In  this  way  the  animal  can  gradually  be  brought 
to  tolerate  doses  of  40  c.c.  or  more. 

Asses. — Asses  are  much  more  susceptible  than  horses  and  react  very  violently. 
In  Marmorek' s  experience,  a  dose  of  5  eg.  of  a  culture  of  which  the  lethal  dose  for 
a  rabbit  is  1  mg.  produces  a  very  severe  reaction.  It  is  well  to  begin  with  less 
virulent  cultures  and  to  increase  the  doses  very  slowly. 

[(7)  Anti-scarlatinal  vaccination. — Gabritchewsky  relying  on  the  analogy  which  he  says 
exists  between  strangles  in  horses  and  scarlatina  in  man  proposed  to  immunize  children 
against  the  latter  with  a  vaccine. 

[The  vaccine  consists  of  a  broth  culture  of  a  streptococcus  isolated  from  a  case  of  scarlet 


VACCINATION  605 

fever.  The  culture  is  sterilized  at  60°  C.  and  centrifuged  :  part  of  the  supernatant  fluid 
is  then  decanted  until  each  cubic  centimetre  contains  0'005  gram  of  dried  organisms. 

[Three  inoculations  are  given.  The  first,  for  children  of  2-10  years,  consists  of  0'5  c.c. 
of  the  vaccine,  the  second  of  O'75-l  c.c.  and  the  third  of  1*5-2  c.c. 

[Inoculation  is  followed  by  a  slight  local  and  general  reaction  :  in  some  cases  by  a 
scarlatiniform  eruption  and  sore  throat.  Gabritchewsky  considers  that  the  latter  symp- 
toms prove  the  specificity  of  the  vaccine. 

[For  various  reasons,  it  is  hardly  likely  that  this  method  of  vaccinating  against  scarlet 
fever  will  prove  to  be  of  any  value.  ] 

[(5)  Vaccines  in  human  streptococcal  infections. — Autogenous  vaccines  are 
now  very  generally  used  in  the  treatment  of  human  streptococcal  infections 
and  with  much  success  especially  if  the  vaccine  treatment  be  combined  with 
the  ordinary  surgical  procedures  and  with  the  use  of  a  specifically  immunized 
serum. 

[Preparation  of  the  vaccine. — Agar  tubes  are  sown  with  material  from  the 
lesion  and  incubated  for  24  hours  at  37°  C.  The  growth  is  then  scraped  off 
with  a  slender  glass  rod  and  made  into  an  emulsion  with  normal  saline  solu- 
tion to  which  0'5  per  cent,  carbolic  acid  has  been  added.  The  emulsion  is 
then  sterilized  either  by  heating  at  60°  C.  for  half  an  hour  or  by  placing  it 
in  the  incubator  for  24  hours  at  37°  C.  The  sterility  must  be  tested  by 
cultivation. 

[Standardization. — The  emulsion  is  then  standardized  either  by  Wright's 
method  (p.  381)  or  by  means  of  a  Thoma-Zeiss  counting  chamber.  If  the 
latter  be  employed  it  will  be  found  convenient  to  use  a  dilute  solution  of 
Azur  II  containing  1  per  cent,  of  commercial  formalin  (to  precipitate  the 
organisms)  as  the  diluent — the  dye  stains  the  organisms  and  so  renders  them 
more  distinct. 

[Method  of  use. — The  vaccine  should  be  inoculated  at  the  earliest  possible 
moment  and  while  it  is  being  prepared  it  is  recommended  that  a  dose 
(lOxlO6)  of  stock  vaccine — prepared  from  a  mixture  of  streptococci  of  the 
variety  which  are  known  commonly  to  cause  the  same  type  of  lesion — should 
be  administered.  The  dose  (10  to  100  millions)  and  the  intervals  between 
the  inoculations  will  vary  according  to  circumstances — "  the  more  acute 
and  the  more  generalized  the  lesion  the  smaller  should  be  the  dose  intro- 
duced "  (Girling  Ball). 

[Sensitized  vaccines. — Sensitized  vaccines  prepared  according  to  Besredka's 
method  (p.  382)  have  recently  been  introduced  for  the  treatment  of  human 
streptococcal  lesions  with  encouraging  results.  As  in  the  case  of  non-sensi- 
tized vaccines  an  autogenous  vaccine,  i.e.  a  vaccine  prepared  from  the  lesion 
to  be  treated,  must  be  used.] 

5.  Serum  therapy. 

[The  serum  therapy  of  streptococcal  infections  is  highly  unsatisfactory.  It 
is  now  more  than  14  years  since  Marmorek  introduced  his  antistreptococcal 
serum  and  though  it  would  appear  in  some  cases  that  the  success  following 
the  use  of  the  serum  has  been  very  striking,  in  the  vast  majority  of  cases  it 
is  far  otherwise. 

[The  difficulties  attending  the  preparation  of  an  antistreptococcal  serum 
are  greatly  enhanced  by  the  fact  that  it  is  still  unknown  whether  the  organisms 
isolated  from  streptococcal  infections  are  all  identical  or  whether  several 
different  species  of  streptococci  exist.  Upon  the  solution  of  this  question 
the  whole  problem  of  streptococcal  serum  therapy  largely  turns. 

[Marmorek  regarded  all  streptococci  as  identical,  and  this  view  was  shared 
by  Neufeld,  and  until  recently  by  Aronson  also.  These  observers  therefore 
prepared  a  monovalent  serum  using  a  single  strain  of  streptococcus  artificially 


606  THE   STREPTOCOCCI   OF   MAN 

increased  in  virulence  by  passage  through  animals.  In  the  laboratory  these 
serums  give  good  results  with  all  streptococci  whatever  their  source  provided 
that  the  virulence  of  the  latter  be  first  raised  by  passage.  But  as  Besredka 
points  out,  a  "  passage  "  streptococcus — whose  properties  have  been  pro- 
foundly altered  by  artificial  means — differs  widely  from  an  organism  which 
has  not  been  subjected  to  treatment  and  whose  pathogenicity  is  that  natural 
to  it,  and  herein  probably  lies  the  explanation  of  the  discrepancies  between 
laboratory  results  with  these  serums  and  clinical  experience. 

[Other  observers  acting  on  the  theory  that  there  are  a  number  of  different 
streptococci  have  prepared  polyvalent  serums.  These  latter  may  be  regarded 
as  of  two  types :  (1)  serums,  as  for  instance  that  of  Tavel,  prepared  by  im- 
munizing animals  with  a  number  of  strains  from  different  clinical  streptococcal 
infections  but  previously  "  exalted  "  by  passage  and  (2)  Besredka's  serum 
which  is  also  prepared  with  a  number  of  different  strains  which  however  have 
not  been  increased  in  virulence  previously  to  being  used  for  immunization.] 

A.  Monovalent  serums. 

Marmorek's  serum. — Marmorek  was  the  first  to  prepare  an  antistreptococcal 
serum  for  therapeutic  purposes. 

Preparation  of  Marmorek's  serum. — Marmorek  obtains  his  serum  from  horses. 
The  animals  are  immunized  in  the  manner  described  above  and  should,  in  order  to 
yield  a  potent  serum,  be  inoculated  with  considerable  doses  of  virulent  cultures, 
each  horse  receiving  at  least  2  litres  of  culture  administered  in  increasing  doses 
over  a  period  of  6—12  months.  An  interval  of  4  weeks  should  elapse  after  the  last 
inoculation  before  the  horse  is  bled  (vide  B.  diphtheria  for  the  technique  of  the 
collection  of  serum). 

Each  inoculation  of  a  living  culture  into  a  horse  for  purposes  of  immunization  is 
followed  by  a  period  of  reaction  during  which  the  blood  is  toxic  but  contains  no 
streptococci.  The  two  following  experiments  illustrate  this.  (1)  A  rabbit  was  inoculated 
with  2  c.c.  of  serum  which  was  collected  during  a  period  of  febrile  reaction  from  an 
already  highly  immunized  horse.  The  rabbit  died  in  a  week.  (2)  Serum  taken  from  a 
horse  in  a  less  advanced  stage  of  immunization  and  a  fortnight  after  the  temperature 
had  become  normal  was  found,  when  inoculated  in  doses  of  O'5-l  c.c.  into  animals 
weighing  1400  grams,  to  kill  rabbits  in  5-10  days. 

The  serum  ceases  to  be  toxic,  and  is  therapeutic  3  weeks  after  the  last  inoculation  ; 
its  therapeutic  properties  are  most  pronounced  at  the  end  of  the  fourth  week  (Mar- 
morek). 

Properties  of  the  serum. — Marmorek's  serum  exhibits  curative,  prophylactic 
and  antitoxic  properties  when  tested  on  rabbits.  It  is  not  bactericidal  in 
vitro  and  has  very  feeble  agglutinating  properties. 

Bactericidal  properties. — Streptococci  grow  in  the  serum  slowly  and  feebly 
as  they  do  in  normal  horse-serum.  Growth  also  takes  place  in  a  mixture 
of  the  serum  and  rabbit-serum  as  in  a  mixture  of  normal  horse-serum  and 
rabbit-serum  and  the  virulence  of  the  organism  is  unimpaired  (Mironoff  : 
Bordet). 

Agglutination. — The  property  of  agglutinating  streptococci  possessed  by 
Marmorek's  serum  is  very  feeble  and  irregular.  To  a  given  volume  of  culture 
emulsion  at  least  one-third  of  that  amount  of  serum  must  be  added  (Bordet). 

Antitoxic  properties.— Using  a  toxin  of  which  1  c.c.  is  fatal  to  rabbits  in 
3  or  4  days  it  is  found  that  if  3-5  c.c.  of  the  serum  be  mixed  with  the  fatal 
dose  the  mixture  is  harmless  on  inoculation  into  a  rabbit. 

Prophylactic  properties. — If  a  rabbit  be  inoculated  under  the  skin  with  2  c.c. 
of  the  serum  and  24  hours  later  with  0*000,001  c.c.  of  a  culture  which  will 
kill  a  control  animal  in  a  dose  of  0-000,000,1  c.c.  the  animal  survives  the 
inoculation.  A  dose  of  serum  equivalent  to  r^ih  of  the  weight  of  an 
animal  protects  it  against  infection. 


SERUM  THERAPY  607 

Curative  properties. — 1  c.c.  of  the  serum  will  save  the  life  of  a  rabbit  inocu- 
lated 3  hours  previously  with  10  times  the  lethal  dose  of  an  exalted  (passage) 
virus  :  5  c.c.  will  in  like  manner  cure  rabbits  inoculated  5  hours  previously  : 
but  in  the  case  of  animals  inoculated  with  the  virus  6  hours  before  the  admini- 
stration of  the  serum,  the  latter  is  powerless  to  prevent  infection.  When  an 
organism  of  ordinary  virulence  for  the  lower  animals  is  used  as  the  test 
[streptococci  taken  direct  from  human  lesions  are  sometimes  non- virulent  for 
animals],  inoculation  of  serum  even  24  or  30  hours  later  leads  to  the  recovery 
of  the  animal. 

According  to  Behring  and  Knorr,  Marmorek,  Aronson  and  others,  if  an  animal  be 
immunized  against  one  strain  of  streptococcus  it  is  equally  immunized  against  related 
strains  and  the  serum  is  efficient  in  both  cases.  This  opinion  would,  however,  not 
seem  to  be  justified  by  clinical  experience  :  thus,  Mery  was  unable  with  Marmorek' s 
serum  to  immunize  rabbits  against  a  streptococcus  isolated  from  the  blood  of  a 
case  of  scarlet  fever  (vide  infra)  and  Courmont  has  proved  that  a  serum  prepared 
according  to  Marmorek' s  directions  is  only  effective  against  the  strain  used  for  its 
preparation  and  not  against  strains  from  other  sources. 

Use  of  Marmorek's  serum  in  practice. — Marmorek's  serum  has  been  used 
in  cases  of  streptococcal  infections  in  man  but  without  any  striking  result. 

In  cases  of  erysipelas  and  puerperal  septiccemia  the  results  have  not  come  up  to 
expectation.  The  serum  is  generally  inoculated  in  doses  of  10  c.c.  repeated,  if 
necessary,  daily  for  a  week  or  more  :  in  serious  cases  20  c.c.  has  been  the  initial 
dose. 

In  scarlet  fever,  Marmorek's  serum  has  yielded  encouraging  but  not  conclusive 
results.  [It  is  conceivable  that  these  results  were  obtained  in  those  cases — which 
are  very  common — in  which  the  streptococci  were  associated  as  a  secondary 
infection.  ] 

In  cases  of  faucial  diphtheria  in  which  streptococci  are  present  as  a  secondary 
infection,  Roux  has  tried  mixing  antidiphtheria  serum  with  the  serum  of  rabbits 
immunized  by  Marchoux  against  streptococci :  the  results  were  not  successful. 
Martin  used  Marmorek's  horse-serum  in  combination  with  antidiphtheria  serum  and 
obtained  rather  more  satisfactory  results. 

In  the  disease  known  as  anasarca  of  the  horse,  caused  by  a  streptococcus  similar 
to  those  found  in  man,  Marmorek's  serum  has  given  good  results  (Nocard  and  others) 
and  has  lowered  the  death-rate  considerably. 

Aronson 's  serum. — Like  Marmorek,  Aronson  regarded  all  streptococci 
occurring  in  human  disease  processes  as  belonging  to  one  and  the  same  species. 
His  investigations  were  carried  out  on  17  strains  of  streptococci. 

Preparation  of  the  serum. — Aronson  increased  the  virulence  of  the  organism  by 
passing  it  through  a  number  of  mice.  In  this  way  a  streptococcus  was  obtained 
which  would  kill  mice  in  a  dose  of  0*000,000,01  c.c.  of  a  glucose-broth  culture. 
Horses  were  then  immunized  with  increasing  doses  of  this  passage  virus. 

The  serum  of  these  animals  is  prophylactic  in  the  case  of  mice  against 
streptococci  of  human  origin  provided  always  that  the  virulence  has  been  raised 
by  passing  it  through  mice.  It  does  not  protect  mice  against  the  streptococcus 
of  strangles  (Chap.  XLII.)  when  first  isolated  from  the  horse  but  is  very  efficient 
against  that  organism  when  it  has  been  passed  through  several  mice.1 

The  prophylactic  properties  of  Aronson's  serum  are  shown  by  the  fact  that  if 
0'0002  c.c.  be  inoculated  into  the  peritoneal  cavity  of  a  mouse  and  24  hours  later 
one  hundred  fatal  doses  of  a  passage  virus  be  inoculated,  the  mouse  survives. 

Aronson's  serum  also  possesses  curative  properties. 

Thus,  if  inoculated  7  hours  after  an  intra-peritoneal  inoculation  of  the  virus  the 
animal  recovers,  and  even  if  the  administration  of  the  serum  be  delayed  for  24  hours 
it  still  leads  to  recovery  in  50  per  cent,  of  the  animals. 

1  Marmorek's  serum  has  very  little  effect  on  a  virus  which  has  been  passed  through 
mice. 


608  THE   STREPTOCOCCI   OF  MAN 

Agglutination.— Aronson's  serum  has  considerable  agglutinating  pro- 
perties for  most  streptococci.  Some  cultures  are  agglutinated  in  a  dilution 
of  1  in  20,000. 

[More  recently  Aronson,  in  immunizing  horses,  has  used  in  addition  to  his 
passage  organism  a  number  of  other  streptococci  which  have  not  been  passed 
through  animals.] 

Moser's  serum. — With  a  view  to  the  preparation  of  an  antiscarlatinal  serum  Moser 
used  a  number  of  streptococci  isolated  from  cases  of  scarlet  fever.  He  immunized 
horses  by  repeatedly  inoculating  them  with  living  cultures  obtained  by  sowing  broth 
with  blood  from  persons  suffering  from  scarlet  fever.  No  attempt  was  made  to  increase 
the  virulence  of  the  organism  by  passage  before  using  it  for  immunization. 

Moser's  serum  agglutinated  .streptococci  isolated  from  cases  of  scarlet  i'ever.  In  the 
laboratory  it  has  some  action,  though  feeble  and  inconstant,  on  streptococci  which  have 
been  passed  through  mice  (Sommerfeld). 

In  the  treatment  of  scarlet  fever  opinions  differ  as  to  its  value.  According  to  some 
observers  it  has  a  very  beneficial  action  on  the  streptococcal  infections  of  the  disease 
(Moser;  Paltauf:  Pospischill).  Others  unfortunately  have  failed  to  secure  these  beneficial 
results  (Moltchanoff,  Baginski,  Czerny)  and  as  a  therapeutic  agent  it  is  now  practically 
discarded.  There  is  "nothing,  from  the  biological  point  of  view,  to  justify  the 
specificity  of  such  a  serum"  (Besredka)  and  it  has  already  been  said  above  that  in 
scarlet  feVer  the  streptococcus  is  merely  a  secondary,  associated,  infection. 

[Andrewes  and  Herder's  serum. — Seeing  that  in  their  experience  the 
Streptococcus  pyogenes  as  denned  by  them  (vide  ante)  is  the  commonest  variety 
in  human  streptococcal  lesions,  Andrewes  and  Horder  prepare  a  specific 
monovalent  antipyogenes  serum  by  using  for  the  inoculation  of  horses  strains 
of  their  Streptococcus  pyogenes.  This  serum  seems  to  give  much  more  satis- 
factory results  than  any  other  antistreptococcal  serum  of  which  they  have 
had  experience  (Andrewes). 

[The  most  beneficial  results  are  obtained  in  practice  by  using  the  serum  in 
conjunction  with  autogenous  vaccines  as  adjuvants  to  the  ordinary  surgical 
procedures.  Thus  in  puerperal  cases — in  which  the  serum  has  been  more 
extensively  used  than  in  any  other  class  of  streptococcal  infection — if  the 
temperature  should  rise  above  normal,  a  dose  (50  c.c.)  of  the  serum  is  admini- 
stered there  and  then  and  a  swab  taken  from  the  interior  of  the  uterus.  The 
uterus  is  then  douched.  Twenty-four  hours  later  a  small  dose  (5  to  10 
millions)  of  a  vaccine  prepared  from  the  organism,  usually  S.  pyogenes,  grown 
from  the  swab  is  given,  followed  later  by  other  doses  of  the  vaccine.  Cases 
treated  thus  at  the  first  sign  of  infection  almost  invariably  do  well  and  do 
not  pass  on  to  septicaemia  (Andrewes).  The  secret  of  success  lies  in  com- 
mencing the  treatment  during  the  earliest  stages  of  infection  and  before 
the  organism  has  passed  beyond  the  uterine  cavity. 

[A  like  procedure  has  been  followed  in  other  forms  of  streptococcal  infec- 
tions— appendicitis,  cellulitis,  arthritis,  etc. — with  distinctly  encouraging 
results  (Girling  Ball). 

[In  streptococcal  infections  caused  by  varieties  of  streptococci  other  than 
S.  pyogenes  similar  treatment  should  be  adopted  and  in  infective  endo- 
carditis the  use  of  a  specifically  immunized  serum  combined  with  an  auto- 
genous vaccine  offers  the  best  hope  of  recovery  (Horder).  It  seems  quite 
likely  as  suggested  by  Horder  that  the  combination  of  a  specifically  immunized 
serum  and  an  autogenous  vaccine  acts  in  a  similar  way  to  the  "  sensitized 
vaccines"  of  Besredka.] 

B.  Polyvalent  serums. 

Convinced  of  the  multiplicity  of  streptococci  and  noting  the  failure  of 
Marmorek's  serum  in  clinical  practice,  various  authors  (Denys,  Van  de  Velde, 
Tavel)  have  prepared,  by  inoculating  animals  with  emulsions  of  different 


SERUM  THERAPY  609 

strains  of  streptococci,  polyvalent  serums  which  they  hoped  would  be  effective 
against  these  different  viruses.  The  results  however  have  been  disappointing. 
Tavel's  serum  is  prepared  in  a  manner  similar  to  that  adopted  by  Marmorek 
but  passage  streptococci  from  various  sources  are  used  for  inoculation.  This  serum 
is  said  to  be  only  prophylactic. 

Besredka's  serum. — This  is  the  anti-streptococcal  serum  now  prepared  at 
the  Pasteur  Institute  in  Paris. 

For  the  preparation  of  the  serum,  Besredka  uses  streptococci  kept  in  a 
medium  consisting  of  equal  parts  of  Martin's  broth  and  horse  serum  heated 
to  56°  C.  for  half-an-hour. 

In  order  to  obtain  a  large  quantity  of  growth  for  purposes  of  inoculation  the 
medium  used  is  agar  contained  in  Roux  bottles  (p.  78)  which  is  watered  with  about 
1  c.c.  of  horse  serum  before  sowing  the  cultures.  An  abundant  growth  is  obtained  in 
24  hours  and  this  is  scraped  off  and  made  into  an  emulsion  with  normal  saline  solution. 

Horses  are  immunized  by  intra-venous  inoculation,  each  inoculation  being  made 
with  six  to  eight  different  strains  of  streptococci  isolated  from  human  lesions  to 
which,  for  purposes  of  standardization,  a  streptococcus  virulent  for  mice  or  rabbits 
is  added.  The  streptococci  are  not  passed  through  animals.  The  immunizing 
process  is  rather  tricky,  each  inoculation  being  marked  by  a  sharp  temperature 
reaction  lasting  about  48  hours.  Occasionally  articular  symptoms  and  inflammatory 
phenomena  develop  10  days  to  a  fortnight  after  the  inoculation :  these  symptoms 
however  rarely  terminate  fatally. 

The  serum  is  not  bactericidal  but  has  considerable  prophylactic  and  curative 
properties.  For  example  a  mouse  inoculated  sub-cutaneously  with  ten  times 
the  fatal  dose  of  streptococci  can  be  saved  by  an  injection  18-24  hours  later 
of  O'OOl  c.c.  of  the  serum  into  the  peritoneal  cavity.  Under  similar  condi- 
tions the  dose  for  a  rabbit  is  T5-2  c.c. 

The  therapeutic  value  of  the  serum  in  human  infections  is  still  subjudice. 

6.   Agglutination. 

Attention  has  already  been  directed  to  the  agglutinating  properties  of  the 
different  serums,  and  from  what  has  been  said  it  will  be  gathered  that  this 
property  is  very  inconstant.  It  not  infrequently  happens  that  a  strain  other 
than  that  used  for  the  preparation  of  a  serum  is  more  powerfully  agglutinated 
than  that  used  for  immunization. 

In  culture,  streptococci  grow  together  and  form  large  granular  masses  so 
that  for  agglutination  tests  an  homogeneous  emulsion  must  be  prepared. 

The  serum  of  persons  suffering  from  streptococcal  infections  has  no 
agglutinating  capacity  for  streptococci. 

7.  Bordet-Geng-ou  reaction. 

[Besredka  expresses  the  opinion  that  it  may  be  possible  by  means  of  the  Bordet- 
Gengou  reaction  to  establish  a  natural  classification  of  the  streptococci.  In  some 
experiments  carried  out  with  Dopter  he  found  that  three  streptococci  from  very 
different  sources  all  had  a  common  fixateur  :  one  of  these  streptococci  was  recovered 
from  a  child  who  had  died  of  septicaemia  another  from  a  case  of  erysipelas  and  the 
third  from  a  child  who  had  died  of  scarlet  fever.  On  the  other  hand  he  several 
times  found  that  two  streptococci  both  isolated  from  the  blood  of  the  heart  of  a 
case  of  scarlet  fever  reacted  differently  to  the  same  fixateur.  ] 

SECTION  IV.— THE   DETECTION  AND   ISOLATION  OF  STREPTOCOCCI. 

(a)  Microscopical  examination. — A  number  of  films  should  be  prepared  with 
the  material  (pus,  blood,  serous  fluid,  etc.)  and  stained,  some  with  carbol- 
thionin  [or  dilute  carbol-fuchsin]  and  others  by  Gram's  method. 

Tissues  for  sections  (erysipelas  skin,  internal  organs,  etc.)  should  be  fixed 

2Q 


610  THE   STREPTOCOCCI   OF  MAN 

in  acid  perchloride  or  absolute  alcohol  and  subsequently  stained  by  Gram's 
method  (double  or  triple  staining,  p.  219). 

(6)  Cultures. — Blood  should  be  sown  in  broth  and  on  agar. 

pus. — To  isolate  streptococci  from  pus  when  other  organisms  are  present^ 
sow  a  little  of  the  material  on  three  agar-slope  tubes  by  the  dilution  method 
(p.  82).  In  this  way  single  colonies  will  be  obtained. 

To  isolate  streptococci  from  a  case  of  erysipelas  :  cleanse  the  skin,  make 
a  prick  with  a  lancet,  and  wipe  away  the  first  drop  or  two  of  blood  with  a 
piece  of  sterile  filter  paper,  then  with  the  thumb  and  index  finger  pinch  up 
the  skin  on  either  side  of  the  puncture,  collect  the  fluid  which  wells  up  in  a 
Pasteur  pipette  and  sow  it  in  broth. 

(c)  Animal  inoculation. — To  determine  the  virulence  of  a  streptococcus 
either  inoculate  the  material  containing  the  organism  directly  into  a  rabbit ; 
or,  better,  make  a  preliminary  culture  in  broth  or  blood-broth,  incubate  at 
37"  C.  for  2  days  and  then  inoculate  a  rabbit. 

[Mice  may,  of  course,  be  used  instead  of  and  are,  in  many  wTays,  more 
convenient  than  rabbits.] 

Meyer  and  Ruppel  sowed  material  containing  streptococci  direct  from  various 
human  diseases  (erysipelas,  scarlet  fever,  inflammatory  conditions,  etc.)  on  to  defibrin- 
ated  human  blood  and  obtained  cultures  which  were  from  the  first  virulent  for 
rabbits  and  mice:  O'Ol  -0'000,001  c.c.  of  these  cultures  was  fatal  to  mice  on  intra- 
peritoneal  inoculation. 

Streptococcus  of  Bonome. 

Bonome  isolated  a  streptococcus  from  the  pus  of  cases  of  cerebro-spinal  meningitis 
and  the  same  organism  has  since  been  found  by  Netter,  Chantemesse,  Bezan9on 
and  Griffon  in  many  cases  of  epidemic  meningitis.  This  streptococcus  apparently 
plays  merely  the  part  of  a  secondary  infection  in  epidemic  meningitis  and  indeed 
has  frequently  been  found  associated  with  the  meningococcus. 

According  to  Netter  this  organism  is  an  attenuated  variety  of  the  pneumococcus  : 
well-marked  differences  however  exist  between  the  two  organisms.  The  strepto- 
coccus meningitidis  appears  to  be  very  closely  related  to  the  streptococcus  mucosu? 
of  Schottmiiller. 

Experimental  inoculation. — White  mice  are  very  susceptible  ;  after  sub-cutaneous 
inoculation  they  die  in  24  hours  of  septicaemia.  'Rabbits  are  rather  less  susceptible 
and  guinea-pigs  are  as  a  rule  refractory  to  sub-cutaneous  inoculation.  Intra-pleural 
inoculation  is  fatal  to  white  rats.  After  several  passages  through  rats  the  organism  no 
longer  forms  chains  but  has  the  morphological  appearance  of  the  pneumococcus  ( Netter ) . 

Microscopical  appearance. — The  streptococcus  of  Bonome  is  an  ovoid  coccus 
which  forms  short  chains  generally  seen  extra-cellularly  and  which  in  pus  and  serum 
cultures  shows  a  capsule. 

It  is  readily  stained  by  the  aniline  dyes  and  is  gram-positive. 

Cultures. — This  streptococcus  grows  easily  on  the  ordinary  media.  Growth  takes 
place  at  20°  C.  and  consequently  the  organism  can  be  grown  on  gelatin.  The 
optimum  temperature  is  37°  C.-380  C.  The  vitality  of  the  streptococcus  of  Bonome 
is  greater  than  that  of  the  pneumococcus  (Bezangon  and  Griffon). 

Broth. — Sown  in  broth  and  incubated  at  37°  C.  the  streptococcus  of  Bonome 
gives  rise  to  a  slight  cloudiness  in  24  hours  and  later  to  a  minimal  deposit. 

Gelatin. — On  gelatin  at  22°  C.  the  organism  gives  rise  to  a  scanty  growth  of  small 
white  opaque  discrete  points.  The  medium  is  not  liquefied. 

Agar. — A  delicate  growth  of  transparent  colonies  similar  to  those  of  the  pneumo- 
coccus is  formed  on  agar  on  incubation  at  37°  C. 

Liquid  rabbit  serum. — The  culture  on  this  medium  is  characteristic  (Bezangon  and 
Griffon).  After  24  hours'  incubation  at  37°  C.  there  is  a  very  slight  cloudiness  of 
the  medium  and  a  little  deposit.  Under  the  microscope  it  will  be  found  that  the 
growth  consists  of  chains  of  variable  length  and  of  diplococci  agglutinated  in  small 
clumps.  The  capsules  are  delicate  and  shrivelled. 

Milk. — Growth  takes  place  and  the  medium  is  sometimes  coagulated  but  often 
unchanged. 

Potato. — No  apparent  growth  on  potato. 


CHAPTER  XLI1. 
STREPTOCOCCI  ANIMALIUM. 

I,  Streptococcus  egui. 
II,  Streptococcus  mammitis  bo  vis,  p.  613. 
Ill,  Micrococcus  mammitis  of  ewes,  p.  615. 

I.  Streptococcus  equi. 

(The  streptococcus  of  strangles.) 

STRANGLES  in  horses  is  caused  by  a  streptococcus  discovered  by  Schiitz, 
which  is  found  also  in  a  number  of  other  though  very  different  clinical  con- 
ditions in  the  horse.  This  organism  has  been  confused  with  the  equine 
pasteurella  which,  as  a  matter  of  fact,  prepares  the  way  for  the  streptococcus 
of  Schiitz. 

It  has  been  pointed  out  already  (Chap.  XXVIII.)  that  in  a  large  number 
of  conditions  originated  by  members  of  the  pasteurella  group  the  original 
infecting  agent  disappears  early  in  the  course  of  the  disease,  so  that  bacterio- 
logical examination  reveals  only  the  presence  of  the  secondary  or  associated 
infection,  in  this  case  the  streptococcus. 

Strangles  is  chiefly  a  disease  of  young  horses  from  1-5  years  old.  The  common 
symptoms  are : — nasal  catarrh,  swelling  and  suppuration  of  the  glands  in  the  sub- 
maxillary  space  (strangles)  and  lymphangitis.  The  lungs  and  pleurae  are  not  infre- 
quently involved  and  the  pleurisy  may  be  of  the  purulent  variety.  Sometimes  the 
disease  may  become  generalized,  in  that  case  there  is  septicaemia  with  metastatic 
abscesses.  The  streptococcus  can  be  found  in  the  discharge  from  the  nose,  in  the 
enlarged  glands,  in  the  abscesses,  in  the  pus  from  the  pleura,  in  the  pulmonary 
lesions,  etc. 

The  streptococcus  equi  though  differing  from  them  in  certain  respects  is 
very  closely  related  to  the  streptococci  hominis.  It  grows  well  in  the  filtrate 
of  a  broth  culture  of  the  streptococci  hominis  (p.  598).  Marmorek's  serum 
has  no  action  upon  it. 

SECTION  I.— EXPERIMENTAL   INOCULATION. 

White  mice. — White  mice  are  the  animals  most  susceptible  to  experi- 
mental infection.  After  subcutaneous  inoculation  an  abscess  forms  at  the 
site  of  inoculation  :  this  is  followed  by  lymphangitis  and  enlargement  of 
the  related  glands,  and  as  in  the  horse,  the  lungs  and  pleurae  may  become 
involved  and  abscesses  may  form  in  the  internal  organs.  The  streptococcus 
is  found  in  pure  culture  in  all  the  lesions  :  in  the  blood  it  only  occurs  in 


612  THE  STREPTOCOCCI  OF  ANIMALS 

small  numbers,  so  that  cultivation  experiments  are  necessary  to  demonstrate 
its  presence.  A  very  highly  virulent  strain  will  cause  a  fatal  septicaemia 
with  only  slight  oedema  at  the  site  of  inoculation. 

Horses. — The  disease  may  also  be  produced  experimentally  in  the  horse. 
Sub-cutaneous  inoculation  leads  to  the  formation  of  an  abscess.  By  rubbing 
the  nasal  fossae  with  a  plug  of  wool  soaked  in  a  culture,  a  clinical  condition 
similar  to  the  spontaneous  disease  is  produced  (Schiitz  and  others).  Intra- 
venous inoculation  is  followed  by  an  interesting  result :  it  leads  merely  to  a 
transitory  illness  but  appears  to  produce  immunity  (Sand  and  Jensen).  [An 
abscess  forms  at  the  site  of  inoculation  which  discharges  externally.  ] 

Mules,  and  more  particularly  asses,  are  less  susceptible  to  the  disease  than 
horses. 

Rabbits  are  even  more  highly  immune  and  to  produce  an  infection  in  these 
animals  the  material  must  be  inoculated  intra-venously.  In  that  case  death 
takes  place  from  septicaemia. 

Guinea-pigs  are  almost  completely  immune  though  it  is  possible  to  set  up 
a  fatal  infection  by  inoculating  large  quantities  of  a  virulent  virus  into  the 
peritoneal  cavity. 

Material  for  inoculation. — For  purposes  of  experimental  inoculation  a 
young  growth  of  the  first  sub-culture  in  broth,  or  pus  from  an  abscess  should 
be  used. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  micro-organism  of  strangles  is  a  coccus  generally  arranged  in  chains 
or  diplococci,  only  rarely  as  isolated  cocci ;  the  chains  may  be  short  con- 
sisting of  three  or  four  cocci  only,  or  long  and  wavy  and  made  up  of  a  large 
number  of  cocci.  The  individual  cocci  measure  0'7-0'9/x  in  diameter  but 
oval-shaped  cocci  with  their  long  axes  transverse  to  the  length  of  the  chain 
are  often  seen.  Encapsulated  cocci  are  not  infrequently  present  in  stained 
films  from  serum  cultures.  A  distinct  capsule  has  been  seen  surrounding 
the  cocci  in  films  made  with  pus  from  the  pleura  in  a  case  of  strangles 
(Besson). 

Staining  reactions. — The  streptococcus  equi  stains  easily  with  the  basic 
aniline  dyes — Kiihne's  blue  or  carbol-thionin.  The  organism  is  gram- 
positive.  Pus  from  a  case  of  strangles  gives  very  pretty  preparations  when 
treated  with  Gram's  stain  and  counter-stained. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  streptococcus  of  strangles  is  an  aerobic  and 
facultative  anaerobic  organism  :  it  grows  best  at  37°  C.  and  only  poorly 
below  20°  C.  According  to  Schiitz  it  only  grows  in  broth  or  on  serum  ;  scanty 
growths  can  at  times  be  obtained  on  agar  and  gelatin  (Nocard,  Sand  and 
Jensen). 

Characters  of  growth.  Broth. — Glycerin-broth  is  the  best  medium.  The 
organism  grows  like  the  streptococcus  of  erysipelas  and  forms  small  white 
flocculi  which  quickly  fall  to  the  bottom  of  the  vessel  leaving  the  medium 
clear. 

Agar. — Cultures  have  been  obtained  on  sloped  agar  (Nocard,  Sand  and 
Jensen).  The  growth  is  more  luxuriant  if  sown  in  deep  stab  culture. 

Besson  had  a  strain  which  grew  fairly  well  on  sloped  agar  for  two  or  three  genera- 
tions ;  the  colonies  were  semi-transparent  and  lenticular-shaped  but  never  exceeded 
in  size  that  of  a  pin's  head. 


BOVINE  MAMMITIS  613 

Gelatin. — Schiitz  and  Poels  failed  to  grow  the  organism  in  stroke  culture  : 
Sand  and  Jensen  succeeded  in  obtaining  cultures  both  on  the  surface  and  in 
stab  culture,  but  the  former  were  very  scanty  indeed. 

With  the  strain  mentioned  above  Besson  secured  a  distinct  growth  on  the  surface 
of  gelatin  at  22°  C.  ;  the  colonies  were  discrete  and  transparent,  and  numbered 
about  ten.  A  sub-culture  sown  on  gelatin  on  the  eighth  day  failed  to  grow. 

Potato. — No  apparent  growth  takes  place  on  this  medium. 

Serum. — On  sloped  serum  the  growth  is  fairly  abundant.  Small,  semi- 
transparent,  lenticular  colonies  at  first  appear  ;  these  soon  become  confluent 
and  form  a  rather  thick,  grey,  iridescent  layer.  The  streptococci  often  show 
a  very  distinct  capsule  on  microscopical  examination. 

Milk. — The  streptococcus  coagulates  milk  in  6-8  days. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 

Viability. — The  streptococcus  of  strangles  is  a  very  delicate  organism. 

Besson  found  that  a  very  actively  growing  culture  in  broth  was  dead  in  12  days. 
When  sown  from  agar  on  to  agar  or  from  gelatin  on  to  gelatin,  sub-cultures  almost 
always  fail.  With  an  organism  which  grew  well  when  first  sown  on  agar  from 
broth,  Besson  obtained  a  scanty  growth  on  the  second  sub-culture  on  agar  but  prac- 
tically none  on  the  third. 

Immunity. — The  immunity  resulting  from  an  attack  of  the  naturally 
acquired  disease  is  of  very  short  duration.  Sand  and  Jensen  succeeded  in 
immunizing  a  horse  by  inoculating  it  intra-venously  with  a  virulent  culture 
and  the  animal  subsequently  proved  to  be  immune  to  intra-nasal  in- 
oculation. 

The  antistreptococcal  serum  of  Aronson  has  proved  effective  as  a  prophy- 
lactic in  experiments  with  the  streptococcus  of  Schiitz,  provided  that  the 
latter  had  undergone  several  passages  through  mice  :  it  is  however  totally 
ineffective  in  the  case  of  an  organism  obtained  direct  from  the  horse  (Aronson). 

II.  Streptococcus  mammitis  bovis. 

(Contagious  mammitis  of  coius.} 

Contagious  mammitis  of  milch  cows  is  due  to  an  infection  with  streptococci 
(Nocard  and  Mollereau)  :  the  organism  may  be  found  in  very  large  numbers 
in  the  milk  of  the  affected  beasts. 

The  disease  is  characterized  by  the  formation  in  the  gland  of  an  indurated  nodule, 
which  may  attain  the  size  of  the  fist  and  even  invade  the  whole  organ.  The  disease 
runs  a  chronic  course  and  does  not  endanger  the  life  of  the  animal :  it  is  spread 
by  the  conveyance  of  the  specific  organism  from  one  cow  to  another  on  the  hands 
of  the  milker. 

The  milk  shows  the  following  characteristic  changes.  Microscopically  it  is  seen 
to  contain  pus  cells  and  numerous  chains  of  cocci.  The  reaction  is  occasionally  acid. 
Sometimes  the  milk  is  normal  in  appearance  at  the  time  of  milking,  but  it  rapidly 
turns  acid  and  coagulates.  If  collected  aseptically  in  sterile  tubes  (p.  201)  and 
kept  at  room  temperature  the  milk  soon  turns  acid  and  clots  while  the  streptococci 
increase  in  number. 


SECTION  I.— EXPERIMENTAL  INOCULATION. 

The  inoculation  of  a  few  drops  of  a  young  culture  or  of  some  infected  milk 
into  the  teats  of  cows  or  goats  produces  a  mammitis  with  all  the  features  of 
the  spontaneous  disease.  Laboratory  animals  are  not  susceptible. 


614 


THE  STREPTOCOCCI   OF  ANIMALS 


SECTION  II.—  MORPHOLOGY. 
1.  Microscopical  appearance. 

The  infecting  organism  in  this  disease  is  a  coccus  about  I/A  in  diameter, 
round  or  somewhat  oval  and  arranged  in  chains.  In  cultures  the  chains  are 
very  long,  often  extending  beyond  the  field  of  the  microscope,  while  in  milk 
and  in  the  affected  tissues  they  are  distinctly  shorter. 


. 
/     .—  •.        :  i          . 

••••- 


•/r   s  =:••;.-..•• 

A     \$?( 

-•'.-'  '-r*\...v "  «  \ 
l-  j     /    -•-/ 


FIG.  287.  —  Streptococcus  mammitis  bo-vis. 
Film  from  a  broth  culture.  Carbol-thionin. 
(Oc.  II,  obj.  TUh,  Reich.) 


FIG.   288.  —  Streptococcus  mammitis  bovis. 
Film  from  the  milk  of  an  infected  cow. 


Staining  reactions.  —  The  streptococcus  mammitis  stains  well  with  the  basic 
aniline  dyes  :  it  stains  badly  by  Gram's  method  and  is  easily  decolourized. 
Films  prepared  with  milk  or  cultures  should  be  stained  with  carbol-thionin 
or  carbol-blue. 

Sections  of  the  gland  are  best  stained  with  carbol-blue  and  tannin  (p.  217). 

2.  Cultural  characteristics. 

Conditions  of  growth.  —  The  streptococcus  is  indifferently  aerobic  ;  the 
ordinary  media  are  quite  suitable  if  slightly  modified  ;  the  optimum  tem- 
perature is  from  35°-37°  C.  and  growth  also  takes  place  at  room  temperature. 

Characters  of  growth.  Milk.  —  The  organism  grows  well  in  milk  which  it 
turns  acid  and  coagulates  in  about  34  hours. 

Broth.  —  The  most  suitable  broth  is  meat  extract  (p.  32)  containing  2-4 
per  cent,  of  glucose  or  lactose.  Incubated  in  this  medium  at  37°  C.  the 
streptococcus  rapidly  forms  a  small  whitish  deposit  sometimes  flocculent 
and  consisting  of  long  chains  of  cocci.  When  left  undisturbed  the  medium 
remains  clear  but  becomes  cloudy  if  gently  shaken. 

The  broth  soon  becomes  very  markedly  acid  and  this  stops  further  growth. 
A  richer  culture  and  one  which  lives  longer  is  obtained  by  adding  2  per  'cent. 
calcium  carbonate  (p.  35)  to  the  broth.  Although  the  organism  dies  in  a 
few  weeks  in  ordinary  media  it  lives  several  months  in  media  containing 
calcium  carbonate. 

Gelatin.  Stab  culture.  —  After  3  or  4  days  small,  whitish,  opaque,  rounded 
colonies  appear,  these  then  run  together  and  form  a  thick  line  of  growth.  The 
gelatin  is  never  liquefied. 

Stroke  culture.  —  Small,  rounded,  translucent  colonies  are  formed  which 
coalesce  to  form  a  pellicle  thicker  at  the  edges  than  in  the  centre. 

Agar.  Serum.  —  On  these  media  the  stroke  cultures  have  the  same  charac- 
teristics as  on  gelatin  but  the  growth  is  more  scanty. 

Potato.  —  The  organism  either  fails  to  grow  altogether  or  produces  a  barely 
visible  film. 


OVINE  MAMMITIS  615 

III.  Micrococcus  mammitis.' 

(The  coccus  of  gangrenous  mammitis  in  ewes.} 

Nocard  has  proved  that  gangrenous  mammitis  in  milking  ewes  is  due  to 
a  coccus  which  has  no  tendency  to  form  chains. 

Bridre  believes  that  this  organism  is  a  normal  inhabitant  of  the  udder  of 
milking  ewes  and  that  mammitis  only  occurs  when  through  some  internal 
lesion  of  the  gland  the  organism  can  penetrate  into  its  tissues. 

Mammitis  in  ewes  is  usually  fatal  in  24-48  hours.  The  mammary  gland  is  hot, 
red,  painful  and  hard ;  the  lesion  then  extends  into  the  sub-cutaneous  cellular  tissue 
of  the  thighs  and  trunk,  while  the  skin  becomes  infiltrated  with  a  serous  oedema 
and  has  an  erysipelatous  appearance ;  later  the  affected  parts  become  gangrenous 
and  the  animal  dies.  The  coccus  is  present  in  large  numbers  in  the  milk,  in  the 
mammary  gland,  and  in  the  blood-stained  fluid  in  the  sub-cutaneous  cellular  tissue 
and  peritoneal  cavity.  The  infection  may  perhaps  be  carried  by  the  milker's  hands  : 
Nocard  certainly  failed  to  cause  infection  by  painting  the  teats  of  healthy  ewes  with 
a  virulent  culture,  but  the  injection  of  a  few  drops  of  the  same  culture  into  the  milk 
ducts  resulted  in  infection  even  though  the  mucous  membrane  was  intact. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

Ewes  readily  contract  the  disease  if  a  few  drops  of  infected  milk  or  of  a 
24-hour  culture  of  the  coccus  be  injected  into  the  teats  or  substance  of  the 
gland. 

Goats  and  laboratory  animals  are  not  susceptible.  In  rabbits  an  abscess 
forms  at  the  site  of  inoculation  and  the  animals  recover. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  micro-organism  of  the  mammitis  of  ewes  is  a  very  small  coccus 
measuring  about  O2//,  in  diameter.  The  cocci  are  arranged  in  pairs,  in  tetrads 
or  in  small  clumps.  They  stain  very  well  with  the  basic  aniline  dyes  and  can 
be  seen  but  with  difficulty  in  unstained  preparations.  They  are  gram- 
positive. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  coccus  of  Nocard  is  indifferently  aerobic.  It 
grows  in  the  ordinary  neutral  or  alkaline  media.  The  optimum  temperature 
lies  between  35°  and  39°  C.  but  it  will  grow  at  the  ordinary  temperature  of 
the  room. 

To  preserve  its  virulence  sub-cultures  should  be  re-sown  daily. 

Like  the  Streptococcus  mammitis  of  cows,  Nocard 's  coccus  remains  alive 
longer  in  broth  containing  calcium  carbonate  than  in  ordinary  broth. 

Characters  of  growth.  Milk. — The  medium  becomes  strongly  acid  and  is 
coagulated  in  24  hours. 

Broth. — Ordinary  broth  or  glucose  broth  become  very  markedly  turbid 
and  a  large  white  precipitate  is  deposited.  A  considerable  amount  of  acid 
is  formed. 

Gelatin.  Stab  cultures.— The  coccus  grows  rapidly  at  20°  C.  Liquefaction 
commences  after  the  second  day  and  is  conical  below  but  the  upper  layers 
are  completely  liquefied  and  turbid. 

1  For  convenience  of  reference  the  organism  causing  mammitis  in  ewes  will  be  con- 
sidered here,  though  morphologically  the  infecting  agent  is  distinct  from  that  of  the 
mammitis  of  milch  cows. 


616  THE  STREPTOCOCCI   OF  ANIMALS 

Agar. — on  agar  slopes  a  thick  spreading  growth  is  formed  which  is  at 
first  white  and  then  somewhat  yellowish. 

Potato. — On  potato  growth  is  always  poor.  A  greyish,  scalloped  film 
forms  which  slowly  acquires  a  yellowish  tint. 

SECTION  III.— VACCINATION. 

Bridre  vaccinated  ewes  by  giving  them  two  inoculations  on  each  side  of 
the  abdomen  of  0*5  c.c.  of  a  broth  culture  attenuated  by  keeping  it  in  the 
hot  incubator  (37°  C.)  for  3  or  4  months.  A  small  abscess  or  slough  often 
formed  at  the  site  of  inoculation  ;  the  animals  were  immunized  in  about  a 
fortnight. 

Bridre  has  shown  that  the  serum  of  hyper-immunized  sheep  has  prophy- 
lactic properties. 


CHAPTER  XLIII. 
STAPHYLOCOCCI  PYOGENETES. 

Introduction. 

Section  I. — The  experimental  disease,  p.  618. 

Section  II. — Morphology,  p.  619. 

1.  Microscopical  appearance  and  staining  reactions,  p.  619.     2.  Cultural  charac- 
teristics, p.  619.     Staphylococcus  pyogenes  aureus,  p.  619.     S.  pyogenes  albus,  p.  620. 
S.  pyogenes  citreus,  p.  620. 
Section  III. — Biological  properties,  p.  620. 

1.  Vitality  and  virulence,  p.  620.     2.  Bio -chemical  reactions,  p.  621.     3.  Toxin, 
p.  622.     4.  Vaccination,  p.  623.    5.  Serum  therapy,  p.  624.     6.  Agglutination,  p.  624. 
Section  IV. — Detection  and  isolation  of  the  staphylococci,  p.  625. 

Diplococcus  crassus,  p.  626. 

SINCE  the  discovery  by  Pasteur  of  the  Staphylococcus  aureus  two  other 
pyogenic  staphylococci  have  been  described,  namely  the  Staphylococcus 
pyogenes  albus  and  the  StapJiylococcus  pyogenes  citreus.  In  their  biological 
properties  these  three  micro-organisms  are  similar,  differing  from  one  another 
only  in  the  colour  of  their  growths.  Rodet  and  Courmont  regard  them 
merely  as  three  races  of  the  same  species  and  this  view  is  probably  correct. 
The  three  organisms  will  therefore  be  described  together  :  the  Staphylococcus 
aureus  will  be  taken  as  the  type  and  the  characteristics  by  which  the  other 
two  varieties  are  differentiated  from  it  will  be  noted  in  the  proper  places. 

The  pyogenic  staphylococci  are  very  widely  distributed  in  nature,  and  are  found 
in  the  air  and  sometimes  in  water,  and  in  man  on  the  skin  and  mucous  membranes,, 
under  the  finger  nails,  and  in  the  alimentary  canal. 

Staphylococci  are  always  present  in  the  mouth :  the  organisms  found  in  this 
situation  and  described  by  Biondi  as  Micrococcus  salivarius  pyogenes,  albus  and 
aureus,  are  identical  with  the  StapJiylococcus  albus  and  aureus  respectively. 

In  human  pathological  lesions  they  are  frequently  found  in  pus,  especially  in 
furunculosis,  osteo-myelitis  (Pasteur),  abscesses  in  various  parts  of  the  body,  etc. 
Occasionally  the  Staphylococcus  enters  the  blood  stream  giving  rise  to  a  purulent 
infection  known  as  pyaemia. 

The  pyogenic  staphylococci  are  found  in  the  lesions  of  suppurative  pleurisy, 
pericarditis  and  peritonitis,  and  also  in  ulcerative  endocarditis  :  they  are  the  cause 
of  some  cases  of  broncho-pneumonia,  of  inflammatory  conditions  of  the  throat, 
bronchitis,  coryza,  etc.  They  are  frequently  associated  with  the  tubercle  bacillus 
in  pleurisy  and  suppurative  meningitis  :  they  complicate  infections  with  the  trico- 
phyton  parasites,  and  are  often  associated  with  the  pneumococcus  in  pneumonia 
and  with  the  diphtheria  bacillus  in  diphtheria  :  they  favour  the  germination  of  the 
spores  of  the  bacillus  of  malignant  oedema  (Besson),  of  the  bacillus  of  hospital  gangrene 
(Vincent),  of  the  bacillus  of  influenza  (Grassberger),  and  of  some  other  organisms. 


618  THE  PYOGENIC  STAPHYLOCOCCI 

Staphylococci  are  found  in  a  very  large  number  of  the  suppurative  conditions 
occurring  in  the  mammalia  and  birds.  The  Staphylococcus  aureus  is  the  infecting 
agent  of  an  osteo- myelitis  occurring  in  young  geese  (Lucet).  The  organism  can 
even  develop  in  fish  and  was  the  cause  of  an  epizootic  which  broke  out  among  the 
gudgeon  in  the  Rhone  (Charrin). 

SECTION  I.— THE  EXPERIMENTAL  DISEASE. 

Among  the  very  numerous  staphylococci  which  can  be  readily  isolated 
from  the  circumambient  media  as  well  as  from  the  various  suppurations 
occurring  in  the  human  body  it  is  but  rarely  that  a  very  virulent  organism 
is  found  ;  in  the  great  majority  of  cases  staphylococci  isolated  from  these 
sources  are  either  very  slightly  virulent  or  totally  avirulent. 

In  the  human  subject. — Garre  produced  boils  by  rubbing  the  skin  energeti- 
cally with  a  piece  of  wool  soaked  in  a  culture  of  Staphylococcus  aureus. 

Rabbits. — The  rabbit  is  the  best  animal  for  purposes  of  experimental 
inoculation. 

Sub-cutaneous  inoculation. — The  sub-cutaneous  inoculation  of  a  few  drops 
of  a  virulent  culture  produces  an  abscess  and  at  the  same  time  a  rise  of  tem- 
perature :  then  the  abscess  points  and  discharges  and  the  animal  is  well 
again,  but  on  rare  occasions  the  organism  may  infect  the  blood  stream  with 
fatal  results. 

Muscatello  and  Ottaviano  using  a  virulent  culture  of  the  Staphylococcus  on  serum- 
broth  produced  a  rapidly  fatal  result  in  rabbits  with  general  dissemination  of  the 
micro-organism  after  sub-cutaneous  inoculation.  No  metastatic  abscesses  were 
formed  but  the  internal  organs  and  especially  the  spleen  showed  lesions  of  necrosis. 

According  to  these  observers,  if  a  culture  of  a  very  virulent  strain  be  inoculated 
death  takes  place  from  toxaemia  before  the  organism  has  had  time  to  become  general- 
ized through  the  tissues. 

Intra-peritoneal  inoculation.— Intra-peritoneal  inoculation  is  more  severe 
than  sub-cutaneous  inoculation  :  it  leads  to  a  rapidly  fatal  septic  peri- 
tonitis. Passage  through  rabbits  increases  the  virulence  of  strains  of  staphy- 
lococci, and  the  organism  can  be  found  in  the  blood  and  internal  organs  of 
animals  that  die. 

Intra-pleural  and  intra-articular  inoculation. — Inoculation  into  the  pleural 
cavity  or  into  a  joint  leads  to  a  purulent  effusion  into  the  cavity,  and  the 
animal  succumbs  in  a  few  days.  If  the  strain  be  very  virulent  it  rapidly 
produces  septicaemia  followed  by  death  in  21—48  hours. 

Intra-venous  inoculation. — Inoculation  of  a  Staphylococcus  into  the  veins 
is  as  a  rule  followed  by  grave  complications.  In  severe  cases  the  organism 
rapidly  invades  the  tissues  and  sets  up  a  condition  of  pyaemia  with  septic 
foci  in  the  internal  organs  and  especially  in  the  kidneys.  Death  takes  place 
in  48  hours  or  more.  With  a  culture  of  increased  virulence,  death  supervenes 
more  quickly  ;  no  metastatic  abscesses  are  formed  but  the  internal  organs 
show  areas  of  necrosis.  Staphylococci  are  found  in  masses  in  the  lumens  of 
the  uriniferous  tubules  of  the  kidney  :  after  the  second  day  no  micro-organisms 
can  be  found  in  the  blood  (Muscatello  and  Ottaviano). 

In  some  cases,  especially  if  lesions  be  artificially  produced  in  the  heart 
beforehand,  inoculation  determines  fatal  ulcerative  or  vegetative  endocarditis 
(Wyssoko witch,  Bibbert,  Bonome). 

Rodet  and  Lannelongue  reproduced  the  lesions  of  osteo-myelitis  by  intra- 
venous inoculation.  This  can  very  easily  be  done  if  a  bone  be  traumatized 
before  inoculation.  In  the  rabbit  it  is  possible  to  produce  an  inflammation  at 
the  junction  of  the  bone  and  epiphysis  similar  to  that  occurring  in  the  human 
subject* 


MORPHOLOGY  619 

If  the  staphylococcus  be  only  slightly  virulent  or  the  dose  injected  be  small, 
a  suppurative  arthritis  is  produced  which  may  either  prove  fatal  or  end  in 
recovery  (Courmont).  Bezancon  and  Griffon  described  a  staphylococcus 
which  invariably  produced  articular  lesions. 

Occasionally  death  does  not  take  place  for  a  long  time  :  in  such  cases 
lesions  of  myelitis  will  be  found  and  the  animal  will  suffer  from  paralysis 
and  convulsions. 

Guinea-pigs,  Rats,  Mice  and  Dogs. — These  animals  are  not  so  constantly 
susceptible  as  rabbits.  In  them,  sub-cutaneous  inoculation  produces  an 
abscess  ;  intra-peritoneal  inoculation  may  terminate  in  a  fatal  septicaemia. 

Geese. — Lucet,  by  inoculating  staphylococci  isolated  from  a  case  of  the 
peculiar  osteo-myelitis  of  young  geese,  was  able  to  reproduce  the  characteristic 
lesions  of  the  disease  in  geese.  Feeding  experiments  and  sub-cutaneous 
inoculation  were  without  result,  but  inoculation  of  broth  cultures  or  of  pus 
from  the  bone  into  the  vein  of  the  wing  proved  fatal  in  3-4  days.  Post 
mortem,  a  multiple  osteo-myelitis  was  found,  and  the  liver  was  very  much 
enlarged  :  the  organism  was  isolated  from  the  bone  marrow,  the  pus  in  the 
bone,  and  from  the  spleen. 


SECTION  II.—  MORPHOLOGY. 
1.  Microscopical  appearance. 

The  staphylococci  are  spherical  cocci  measuring  O6-1//.  in  diameter,  non- 
motile,  generally  arranged  in  irregular-shaped  masses  of  five  to  thirty  cocci 
which  are  often  compared  to  bunches  of  grapes, 
and  rarely   occurring  singly  or  in  pairs  or  in  *,•„•* 

short  chains  composed  of  quite  a  few  cocci.  •£•„, 

Staining  methods.  —  The  staphylococci  stain 

readily  with  the  basic  aniline   dyes  and  are     Jf*.         m  fy         „•**; 
gram-positive.  *£*?$V         *X        %*«* 

These  characteristics  are  common  to  all  varie-       "/•**«>        •§  .• 


ties  of  the  pyogenic  staphylococci.  v*    *          ., 

F 

2.  Cultural  characteristics.  .;,      • 

Physical  conditions.  —  The  staphylococci  grow  :•*  ^    ..^.  0     n 

at  any  temperature  between   10°   and   44°  C.,  *&    :ff  ' 

on  all  culture  media,  aerobically  as  well  as  FlQ,28Q^staphylococcuspyogenes 
anaerobically  :  the  optimum  temperature  is  aureus.  Film  from  a  broth  culture. 
about  35-37°  C.  The  most  favourable  tern-  ^h^SlT1^01^  (0c<m'obj' 
perature  for  pigment  production  lies  between 

20°  and  25°  C.  ;  the  colour  does  not  appear  when  the  cultures  are  grown 
in  vacuo. 

Staphylococcus  pyogenes  aureus. 

Broth.  —  At  37°  C.  the  medium  becomes  cloudy  in  12-24  hours,  after  which 
an  abundant  white  precipitate  is  thrown  down  but  the  broth  still  remains 
cloudy.  Later  on,  the  precipitate  assumes  a  yellowish  tint  and  may  become 
bright  orange  in  colour.  Sometimes  the  pigment  is  slow  in  making  its  appear- 
ance and  may  never  be  very  marked.  In  old  cultures,  the  Staphylococcus 
aureus  often  loses  its  power  of  producing  pigment  and  then  cannot  be  dis- 
tinguished from  the  Staphylococcus  albus. 

Gelatin.  Stab  culture.  —  At  20°  C.  in  24-36  hours  a  granular  growth  appears 
along  the  line  of  sowing  :  towards  the  fifth  day  it  forms  a  funnel-shaped 
liquefaction  filled  with  a  turbid  liquid  and  at  the  bottom  a  yellowish-  white 


620 


THE  PYOGENIC  STAPHYLOCOCCI 


precipitate  :  the  funnel  of  liquefaction  subsequently  enlarges  and  reaches 
the  sides  of  the  tube  and  little  by  little  becomes  cylindrical,  but  it  rarely 
extends  to  the  bottom  of  the  tube.  Some  strains  of  the 
Staphylococcus  aureus  liquefy  gelatin  much  more  slowly 
than  others.  One  of  these  strains  which  in  every  other 
respect  resembled  the  Staphylococcus  aureus  did  not  com- 
mence to  liquefy  gelatin  until  it  had  been  incubated  at 
20°  C.  for  a  fortnight,  and  even  then  the  liquefaction  was 
always  minimal  in  amount  (Besson). 

Single  colonies. — After  incubating  for  2-4  days  at  20°  C. 
small  greyish  circular  colonies  with  a  yellow  centre  appear : 
a  little  later  an  annular  liquefaction  occurs  around  these 
colonies,  which  extends  more  or  less  rapidly.  Yellow  flakes 
can  be  seen  swimming  in  the  cloudy  liquid. 

Agar.  Coagulated  serum.- At  37°  C.  numerous  white 
rounded  colonies  appear  in  24  hours  along  the  line  of  in- 
oculation ;  these  rapidly  coalesce  to  form  a  more  or  less 
shiny  moist  broad  band,  and  the  growth  soon  acquires  a 
colour  varying  between  a  dirty  yellow  and  a  bright  orange 
yellow.  Sometimes  the  colour  does  not  appear  until  about 
the  eighth  or  tenth  day. 

Potato. — It  is  on  this  medium  that  the  Staphylococcus 
aureus  produces  the  most  intense  colour.    Towards  the  second 
or  fourth  day  at  37°  C.  the  growth  forms 
a  more  or  less  bright  yellow  thick  layer. 
Milk. — Growth  rapidly  leads  to  coagu- 
g3n!tab  Culture  in   lation  of  the  medium. 

Staphylococcus  pyogenes  albus. 

This  organism  has  the  same  characteristics  as  the  Staphylo- 
coccus aureus  except  that  the  growths  are  always  white.  On 
agar  the  colour  is  dull  white  like  porcelain.  It  often 
liquefies  gelatin  more  slowly  than  the  Staphylococcus  aureus 
[and  Gordon  has  shown  that  some  strains  fail  to  liquefy 
gelatin.  The  larger  number  of  the  strains  examined  by  Gordon  did  not  clot 
milk]. 

Staphylococcus  pyogenes  citreus. 

Here  again,  the  characteristics  are  the  same  as  those  of  the  Staphylococcus 
aureus  save  that  the  colour  of  the  cultures  is  a  citron  yellow.  [Two  out  of 
the  three  strains  examined  by  Gordon  did  not  clot  milk.] 


coccus  pyogenes  aureus. 


FIG.  291 . — Staphylo- 
coccus pyogenes  aureus. 
Surface  colony  on  a 
gelatin  plate  (5  days). 
xlO. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Viability.    Virulence. 

Viability. — The  staphylococci  do  not  form  spores  :  but  retain  their  vitality 
in  culture  for  a  very  long  time.  In  broth  they  will  still  be  found  living  after 
the  lapse  of  a  year  and  on  gelatin  after  an  even  longer  time. 

Cultures  of  staphylococci  are  easily  killed  by  heat :  e.g.  exposure  to  a 
temperature  of  55°  C.  for  24  hours,  or  of  80°  C.  for  15  minutes  is  sufficient  to 
sterilize  them.  In  dried  pus  or  other  albuminous  material  staphylococci 
will  resist  the  action  of  steam  at  100°  C.  for  several  minutes. 

Staphylococci  in  culture  are  very  susceptible  to  the  action  of  antiseptics 
but  when  mixed  with  albuminoid  substances  are  much  more  resistant. 


BIOLOGICAL  PROPERTIES 


621 


Virulence. — The  virulence  of  the  staphylococci  is  subject  to  variation  which 
cannot  be  anticipated.  As  a  rule,  virulence  is  markedly  lowered  in  old 
cultures,  and  to  maintain  it,  it  is  necessary  to  sow  sub-cultures  every  5  or  6 
days  and  to  pass  the  culture  through  a  rabbit  from  time  to  time  by  intra- 
peritoneal  inoculation. 


FIG.  2Q2.—Staphylo- 
coccus  aureus.  Surface 
culture  on  agar  (5  days). 


FIG.  2Q3.—Staphylo- 
coccus  albus.  Surface 
culture  on  agar  (5  days). 


FIG.  294—Staphylo- 
coccus  citreus.  Surface 
culture  on  agar  (5  days). 


Staphylococci  recovered  from  the  circumambient  media  are  often  avirulent ; 
sometimes  even  the  staphylococcus  isolated  from  a  septic  infection  in  man 
is  absolutely  devoid  of  virulence  for  laboratory  animals. 

The  addition  of  glucose  to  culture  media  increases  the  virulence  of  staphy- 
lococci (Budjwid,  Nicholas). 

2.  Bio-chemical  reactions. 

[Gordon  from  an  investigation  of  a  number  of  staphylococci  obtained  from 
various  sources  (air,  skin,  saliva,  scurf,  urine,  sputum  and  pus)  showed  that 
"  comparison  between  various  staphylococci  in  regard  to  nine  selected  actions 
has  revealed  differences  not  merely  of  degree,  but  of  kind,  and  has  shown 
that  a  differentiation  far  more  elaborate  than  has  yet  been  supposed  to  exist 
naturally  obtains  amongst  staphylococci." 

[The  tests  used  by  Gordon  were  as  follows : — 

1.  The  action    on   gelatin    (12    per   cent.)    with   regard   to   liquefaction   when 
incubated  for  one  week  at  22°  C. 

2.  The  clotting  of  litmus  milk  when  incubated  for  one  week  at  37°  C. 

3.  The  peptonization  of  milk  under  the  same  conditions. 

4.  The  reduction  of  nitrate  to  nitrite  during  incubation  at  37°  C.  for  three  days. 

5.  The  reduction  of  neutral  red  in  a  broth  medium  when  incubated  at  27°  C. 
anaerobically  for  two  days. 

6.  The  production  of  acid  in  a  slightly  alkaline  litmus  broth  containing  1  per 
cent,  lactose  when  incubated  at  37°  C.  for  one  week. 


622  THE  PYOGENIC  STAPHYLOCOCCI 

7.  Similar  conditions,  but  with  maltose. 

8.  With  glycerin. 

9.  With  mannite. 

[Though  the  experiments  were  not  sufficiently  extensive  to  allow  of  a 
classification  of  staphylococci  they  show  quite  clearly  that  the  generally 
accepted  grouping  into  three  types  depending  upon  the  colour  of  the  growth, 
on  an  agar  medium  is  insufficient.] 

3.  Toxin. 

In  cultures  the  staphylococcus  produces  fatty  acids  from  sugars  :  it  con- 
verts lactose  into  lactic  acid  and  under  certain  conditions  produces  acetic, 
valerianic,  butyric  and  propionic  acids.  Cultures  also  soon  give  off  a  musty 
smell  if  kept. 

Staphylococci  produce  a  little  indol.  The  property  of  liquefying  gelatin 
is  due  to  the  elaboration  of  diastases  which  also  peptonize  the  white  of  egg. 
The  cultures  also  contain  soluble  toxins. 

I.  De  Christmas  filtered  a  broth  culture  of  the  Staphylococcus  aureus  through 
a  Chamberland  bougie  and  precipitated  the  filtrate  with  4  or  5  volumes  of 
strong  alcohol.     The  precipitate  was  poured  on  to  a  filter  paper,  washed 
with  alcohol  and  dissolved  with  water.     The  solution  thus  obtained  has 
some  power  of  producing  inflammation,  and  injected  into  the  anterior  chamber 
of  the  eye  of  a  rabbit  sets  up  a  slight  degree  of  suppuration. 

II.  Leber  extracted  from  cultures  a  crystallizable  substance  soluble  in 
alcohol,  which  has  marked  properties  of  producing  inflammation,  suppura- 
tion and  even  necrosis  of  the  tissues  into  which  it  is  injected.     Leber  calls 
this  substance  Pklogosine. 

III.  Rodet  and  Courmont  investigated  the  toxic  products  of  the  Staphylo- 
coccus pyogenes  more  thoroughly. 

(a)  Broth  cultures  incubated  for  20  days  at  35°  C.  were  heated  for  24  hours 
at  55°  C.  to  kill  the  micro-organisms  and  then  filtered  through  paper.  The 
filtrate  was  feebly  toxic  for  dogs  and  rabbits. 

In  dogs  symptoms  of  poisoning  were  seen  after  the  inoculation  of  1  -3  c.c.  per  kg. 
of  body  weight,  but  death  only  occurred  rapidly  (at  the  earliest  in  17  hours)  on  the 
inoculation  of  a  formidable  dose  (35  c.c.  per  kg.)  into  the  jugular  vein.  Under  these 
conditions  it  produced  a  fall  of  temperature,  sickness,  convulsions  and  tremors  and 
tended  to  stop  the  heart  and  respiration. 

Rabbits  are  still  less  susceptible.  A  rabbit  weighing  1900  grams  inoculated  with 
10  c.c.  of  heated  culture  only  died  at  the  end  of  6  days  after  having  lost  flesh  and 
weight. 

The  toxin  is  very  unstable  and  rapidly  loses  its  properties  on  keeping. 

(6)  A  culture  20  days  old  filtered  through  a  Chamberland  bougie  is  even 
less  toxic.  After  the  inoculation  of  doses  as  large  as  10  and  15  c.c.  into  the 
veins  of  rabbits  weighing  2  kg.  the  animals  only  showed  a  transitory  rise 
of  temperature  without  any  loss  of  weight. 

(c)  Cultures  20  days  old  were  decanted  and  the  clear  liquid  filtered. through 
several  folds  of  paper.  The  filtrate  was  precipitated  with  four  times  its 
weight  of  strong  alcohol,  and  the  precipitate  washed  with  alcohol,  dried  and 
dissolved  in  water. 

1.  The  extract  so  obtained  was  only  slightly  toxic. 

To  kill  a  dog  weighing  6  kg.  in  2  hours  it  was  necessary  to  inject  into  the  veins  a 
quantity  corresponding  to  260  c.c.  of  culture.  The  symptoms  were  dyspnoea, 
Cheyne-Stokes'  breathing,  sub-normal  temperature,  tremors,  convulsions  and 
spasms. 

The  rabbit  is  more  resistant:  it  does  not  succumb  to  doses  corresponding  to 


STAPHYLOLYSIX  623 

50  c.c.  of  culture.     An  animal  inoculated  with  the  precipitate  recovered  from 
140  c.c.  of  culture  survived  for  a  week. 

2.  The  alcoholic  solution  was  separated  by  filtration  from  the  albuminoid 
precipitate  and  evaporated  in  vacuo  ;  it  gave  a  residue  which  was  dissolved 
in  water. 

Two  dogs  weighing  9  kg.  each  did  not  succumb  after  the  intra- venous  inoculation 
of  doses  of  this  solution  corresponding  to  200  c.c.  and  500  c.c.  of  culture.  A  dog 
weighing  10  kg.  succumbed  to  an  inoculation  representing  210  c.c.  of  culture  after 
suffering  from  a  generalized  anaesthesia,  loss  of  reflexes  and  finally  stoppage  of  the 
heart  and  respiration. 

The  rabbit  is  less  susceptible.  It  never  succumbed  rapidly  to  the  inoculation 
of  the  material  soluble  in  alcohol.  One  animal  survived  the  inoculation  of  a  dose 
representing  85  c.c.  of  culture  for  20  days. 

The  authors  concluded  that  the  substances  soluble  in  alcohol  (toxin  2)  on 
the  one  hand  and  those  insoluble  in  alcohol  (toxin  1)  on  the  other  separately 
injected  are  more  toxic  than  the  mixture  :  that  these  two  groups  of  sub- 
stances are  in  fact  antagonistic  to  and  partially  neutralize  each  9ther.  The 
feeble  toxicity  of  the  products  obtained  by  Rodet  and  Courmont  is  of  con- 
siderable interest  and  the  experiments  are  worth  repeating. 

IV.  Mosny  and  Marcano  obtained  cultures  in  broth  which  after  nitration 
killed  rabbits  in  a  few  seconds  when  inoculated  intra-venously  in  doses  of 
10  c.c.  In  doses  of  1-2  c.c.  the  nitrate  produces  cachexia  terminating  in 
death  in  5-6  weeks.  Animals  which  survive  the  inoculation  of  toxin  never 
exhibit  any  immunity  against  the  Staphylococcus  pyogenes. 

Leucocidine. — Van  de  Velde  by  inoculating  a  culture  of  the  staphylococcus 
into  the  pleural  cavity  of  a  rabbit  obtained  an  exudate  rich  in  degenerated 
white  cells.  This  exudate  mixed  with  normal  leucocytes  rapidly  destroyed 
the  latter.  It  contained  a  substance,  leucocidine,  which  behaved  as  a  soluble 
ferment  and  was  destructive  to  white  cells.  This  substance  is  not  a  product 
of  reaction  of  the  organism  because  it  is  produced  in  cultures  (Bail). 

Staphylolysin. — Neisser  and  Wechsberg  have  demonstrated  the  presence  of 
an  hsemolysin  in  cultures  of  the  staphylococcus.  If  a  drop  of  rabbit's  blood 
be  added  to  a  culture  of  the  staphylococcus  in  broth  (either  filtered  or  un- 
filtered)  it  will  be  found  that  after  standing  in  the  ice-chest  for  a  few  hours 
the  blood  is  completely  hsemolyzed. 

The  best  method  of  preparing  the  hsemolysin  is  as  follows : — Sow  a  distinctly 
alkaline  broth  (add  one-third  of  the  quantity  of  alkali  necessary  to  make  the  broth 
alkaline  to  phenol-phthalei'n)  with  a  staphylococcus  and  incubate  for  12  days.  Filter 
and  add  a  little  carbolized  glycerin  to  the  filtrate  to  ensure  its  keeping.  The  filtrate 
must  also  be  kept  in  the  ice-chest,  as  the  haemolysin  is  destroyed  in  a  few  days  at  the 
ordinary  temperature  of  the  laboratory  and  much  more  rapidly  at  48—56°  C.  Not 
all  strains  of  staphylococci  produce  an  hsemolysin. :  non-pathogenic  staphylococci 
are  incapable  of  hsemolyzing  blood  (Otto). 

Small  doses  of  haemolysin  inoculated  into  rabbits  sub-cutaneously  cause  a  rise 
of  temperature  and  induration  at  the  site  of  inoculation.  If  the  inoculations  be 
repeated  the  serum  becomes  anti-hsemolytic. 

Normal  human  serum  and  normal  horse  serum  both  possess  anti-haemolytic  pro- 
perties. 

4.  Vaccination. 

[Human  vaccination  in  staphylococcal  infections. — Since  their  introduction 
by  Wright  the  inoculation  of  killed  organisms  has  been  extensively  adopted 
and  with  much  success  in  the  prophylaxis  and  treatment  of  human  staphy- 
lococcal infections  and  especially  of  furunculosis,  carbuncles,  acne,  sycosis, 
chronic  bronchitis,  catarrh  of  the  upper  respiratory  passages,  etc. 

[The  organism  must  be  isolated  from  the  particular  lesion  it  is  proposed 


624  THE  PYOGENIC  STAPHYLOCOCCI 

to  treat  and  the  vaccine  prepared  according  to  the  method  described  for 
the  preparation  of  streptococcal  vaccines  (p.  605).  A  carbolic  vaccine  appears 
to  give  better  results  than  an  heated  vaccine  and  the  emulsion  should  be  of 
such  an  opacity  that  quarter-inch  type  can  be  read  through  it  when  contained 
in  a  6  X  f  in.  tube.  If  a  thicker  emulsion  be  made  the  organisms  may  survive 
the  action  of  the  carbolic  acid  for  several  days. 

[For  the  treatment  of  staphylococcal  infections  an  initial  dose  of  250  X 106 
organisms  may  be  inoculated  and  the  dose  repeated  at  intervals  of  five  to 
seven  days.  If  necessary  the  dose  may  be  increased  as  the  treatment 
proceeds  but  care  must  be  taken  that  too  large  a  dose  be  not  inoculated 
otherwise  the  beneficial  results  will  not  be  obtained.] 

5.  Serum  therapy. 

About  this  branch  of  the  subject  there  is  still  much  to  learn.  The  experi- 
ments are  few  in  number  and  have  not  infrequently  yielded  contradictory 
results. 

Mosny  and  Marcano  failed  in  their  attempts  to  vaccinate  rabbits  by  inocu- 
lating them  with  small  doses  of  an  active  toxin. 

According  to  Courmont  only  toxins  soluble  in  alcohol  possess  vaccinating 
properties ;  the  substances  precipitated  by  alcohol  on  the  other  hand  pre- 
dispose to  infection.  By  injecting  the  soluble  toxin  prepared  by  the  methods 
described  above,  Courmont  has  been  able  to  obtain  some  degree  of 
immunity.  The  serum  of  animals  so  treated  appears  to  attenuate  the  viru- 
lence of  the  staphylococcus.  Control  experiments  by  Tavel  have  failed  to 
confirm  the  researches  of  Courmont. 

Viquerat  and  Kosc,  and  Parascandolo  obtained  a  serum  which  is  both 
prophylactic  and  therapeutic.  This  serum,  prepared  by  inoculating  into 
animals  virulent  cultures  in  sugar  broth  which  have  been  sterilized  by  the 
addition  of  5  per  cent,  of  carbolic  acid,  is  said  to  be  both  antitoxic  and 
bactericidal. 

Capman  inoculated  rabbits  and  dogs  with  a  filtered  culture  of  a  staphy- 
lococcus grown  in  1  per  cent,  peptone  broth  and  incubated  at  37°  C.  for  3 
weeks.  After  several  injections  of  toxin  the  animal  was  left  alone  for  a  fort- 
night or  3  weeks  and  then  bled.  This  blood  was  both  bactericidal  and 
antitoxic,  and  inoculated  into  guinea-pigs  and  rabbits  protected  them  from 
and  even  cured  them  of  a  staphylococcal  infection. 

Paltchikowsky  immunized  a  horse  with  repeated  sub-cutaneous  inocula- 
tions of  a  culture  of  the  Staphylococcus  aureus,  and  obtained  a  serum  which 
when  injected  sub-cutaneously  protected  the  animal  against  twice  the  fatal 
dose  of  staphylococci  injected  into  the  veins. 

According  to  Proscher  only  the  inoculation  of  living  staphylococci  leads 
to  the  production  of  an  active  antistaphylococcal  serum.  Proscher  inocu- 
lated goats  and  horses  with  a  very  virulent  staphylococcus  recovered  from  a 
boil  on  the  lip.  A  goat  which  in  the  space  of  a  month  was  inoculated  with 
7  agar  cultures  and  30  broth  cultures  of  this  staphylococcus  yielded  a  serum 
of  which  1-3  c.c.  inoculated  under  the  skin  protected  rabbits  against  five 
times  the  fatal  dose  of  the  virus  inoculated  into  the  veins. 

6.  Agglutination. 

Kolb  and  Otto  prepared  a  serum  possessing  marked  agglutinating  pro- 
perties. This  serum  (prepared  with  a  staphylococcus  isolated  from  the 
human  subject)  has  powerful  agglutinating  properties  for  the  Staphylococcus 
pyogenes  but  is  without  any  action  on  saprophytic  non-pathogenic  staphy- 
lococci (this  test  may  be  applied  in  the  identification  of  the  organism). 


ISOLATION   OF  THE   STAPHYLOCOCCI  625 

The  serum  obtained  by  Proscher  (vide  ante)  agglutinates  virulent  staphy- 
lococci  in  high  dilutions  (1-2500). 

The  serum  of  a  person  suffering  from  a  staphylococcal  infection  has  no 
agglutinating  properties. 


SECTION  IV.— THE  DETECTION,  ISOLATION  AND  IDENTIFICATION 
OF  THE  STAPHYLOCOCCI. 

In  suspected  staphylococcal  infections  the  pus,  blood  and  exudates  should 
be  examined. 

(a)  Microscopical  examination.— Films  and  smears  made  with  blood,  pus, 
etc.  should  be  stained  : 

1.  Some,  with  one  of  the  solutions  containing  carbolic  acid — (Kiihne's 
blue,  carbol-thionin,  or  dilute  carbol-fuchsin). 

2.  Others,  by  Gram's  method.     The  staphylococci  are  gram-positive  and 


«""•>  V$,       -^j 

** 


FIG.  295. — Staphylococcus  aureus.     Pysemic  abscess  of  the  lung.    Gram's 
stain  and  eosin.    (Oc.  2,  obj.  ,Vth.  Zeiss.) 

by  double  staining,  using  eosin  for  the  ground- work,  very  pretty  preparations 
are  obtained. 

(b)  Cultures. — The  Staphylococcus  may  not  be  present  in  pure  culture  in 
the  original  material,  and  sometimes  more  than  one  variety  of  Staphylococcus 
may  be  present,  so  that  methods  of  isolating  the  organisms  in  pure  culture 
will  have  to  be  adopted. 

1.  Sow  a  drop  of  the  material  in  a  tube  of  broth  (this  will  give  a  culture  of 
all  the  organisms  present). 

2.  To  isolate  the  different  organisms  sow  a  loopful  of  the  material  on  the 
surfaces  of  three  agar  tubes  without  recharging  the  needle,  according  to 
the  method  described  at  p.  82.     In  this  way  single  colonies  are  obtained 
which  it  is  quite  easy  to  differentiate. 

Gelatin  plates  may  also  be  sown,  but  this  method  is  liable  to  lead  to  error, 
as  some  organisms  such  as  the  pneumococcus  and  the  streptococcus  may 
pass  unnoticed. 

Note. — In  investigating  the  organisms  present  in  a  septic  infection  it  must  be 
remembered  that  staphylococci  are  quite  commonly  found  on  the  surface  of  the 
skin,  so  that  every  precaution  must  be  taken  to  eliminate  this  possible  source  of 

2R 


626  DIPLOCOCCUS  CRASSUS 

error  (Chap.  XII.).'    It  is  well  to  cauterize  a  small  area  of  the  surface  through  which 
to  pass  the  needle  or  pipette  for  the  collection  of  the  material. 

(c)  Inoculations. — To  ascertain  the  virulence  of  cultures,  rabbits  should 
be  inoculated  both  sub-cutaneously  and  also  intra-venously  into  the  ear 
vein.  . 

Diplococcus  crassus. 

The  organism  isolated  by  Jaeger  from  cases  of  cerebro-spinal  meningitis  and 
identified  by  Jaeger  and  Huebner  with  the  Meningococcus  (Chap.  XL VII.)  is  clearly 
a  different  organism  from  the  latter  and  is  identical  with  the  Diplococcus  crassus. 
It  is  a  saprophyte  inhabiting  the  pharynx  but  may  be  found  in  association  with  the 
Meningococcus  and  even  with  the  tubercle  bacillus  in  cases  of  meningitis.  It  differs 
mainly  from  the  Meningococcus  in  the  following  particulars  : 

1.  Staining  reactions. — The  Diplococcus  crassus  is  gram-positive. 

2.  Cultures. — It  grows  on  ordinary  media  at  20°  C.  and  above. 

3.  Fermentation  reactions. — It  ferments  nearly  all  the  sugars  notably  Isevulose, 
galactose,  lactose  and  saccharose  on  all  of  which  the  Meningococcus  has  no  action. 

4.  Agglutination. — The  Diplococcus  crassus  is  not,  as  a  rule,  agglutinated  by  an 
antimeningococcal    serum :     occasionally   however   agglutination   is   noticed   with 
dilutions  of  1  in  25  and  1  in  50.     But  this  (is  a  group-agglutination  similar  tojthat 
which  occurs  with  the  Gonococcus  and  referred  to  later  (Chap.  XLVIL). 

An  anticrassus  serum  has  no  agglutinating  action  on  the  Meningococcus  (Wasser- 
mann). 

The  organism  described  by  Lepierre  as  a  Meningococcus  exhibits  all  the  charac- 
teristics of  the  Jaeger- Huebner  coccus  and  is  apparently  identical  with  that  organism  : 
Lepierre  found  that  the  virulence  was  increased  by  passage  through  the  peritoneal 
cavities  of  rabbits. 


CHAPTER  XLIV. 
THE  ENTEROCOCCUS. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  627. 

Section  II. — Morphology  and  biological  properties,  p.  628. 

Section  III. — Detection  and  isolation  of  the  enterococcus,  p.  629. 

SEVERAL  years  ago  Escherich,  Tavel,  Eguet,  recorded  the  finding  of 
encapsulated  streptococci  in  the  intestines  of  new-born  children,  and  in 
1894-7  Besson  described  a  new  "  encapsulated  streptococcus  "  which  he  had 
isolated  from  two  cases  of  post-typhoid  suppuration  (purulent  pleurisy, 
multiple  suppurative  arthritis).  These  organisms  are  identical  with  the 
Enterococcus,  of  which  Thiercelin  gave  the  classical  description. 

The  Enterococcus  is  a  saprophytic  micro-organism  which  under  certain 
conditions  may  become  pathogenic  :  it  is  widely  distributed  and  occurs  in 
the  alimentary  canal,  in  the  mouth,  nose  and  pharynx,  on  the  skin,  in  the 
vagina,  etc. 

It  plays  an  important  role  in  the  enteritis  of  children  and  adults  as  well 
as  in  infections  of  the  liver  ;  it  is  also  the  cause  of  some  cases  of  meningitis, 
broncho-pneumonia,  etc.,  as  well  as  of  some  of  the  complications  of  enteric 
fever  and  tuberculosis. 

Rosenthal  has  described  a  disease  of  the  lungs  due  to  this  organism,  characterized 
by  a  pseudo-lobar  broncho-pneumonia  accompanied  with  severe  general  symptoms, 
and  followed  by  a  condition  of  cachexia  which  may  lead  to  it  being  mistaken  for 
pulmonary  tuberculosis. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

The  virulence  of  the  enterococcus  is  very  variable  :  often  the  organism 
is  totally  devoid  of  pathogenic  power. 

Mice  are  very  susceptible  to  infection ;  sub-cutaneous  inoculation  of  a 
virulent  strain  leads  to  death  from  septicaemia  in  24r-48  hours.  The  organism 
may  be  found  in  the  blood,  internal  organs  and  in  the  diarrhosal  contents  of 
the  intestine. 

Rabbits  are  less  susceptible  though  intra-venous  inoculation  of  a  virulent 
strain  produces  a  fatal  septicaemia  :  intestinal  lesions  are  always  present 
post  mortem.  The  enterococcus  isolated  by  Besson  from  one  of  his  cases  of 
post-typhoid  suppuration  produced  multiple  suppurative  arthritis  on  intra- 
venous inoculation  into  rabbits  and  killed  the  animals  in  a  fortnight  to  3 
weeks. 


628  THE  ENTEROCOCCUS 

Guinea-pigs  again  are  less  susceptible  than  mice  but  they  also  die  of 
cachexia  several  weeks  after  being  inoculated  with  an  enterococcus  the 
virulence  of  which  has  been  raised  by  passage  through  mice  (Thiercelin). 
According  to  Eosenthal  lesions  are  found  in  the  intestines  of  guinea-pigs 
killed  by  intra- venous  inoculation. 

The  saprophytic  enterococcus  becomes  virulent  after  passage  through 
animals  (Thiercelin  and  Jouhaud).  These  observers  inoculated  a  large  dose 
of  a  broth  culture  of  the  saprophyte  beneath  the  skin  of  a  rabbit :  an  abscess 
formed  at  the  site  of  inoculation  and  the  enterococcus  was  found  in  the  pus. 
When  this  pus  was  inoculated  into  mice  the  latter  died  of  septicaemia  in  2  or 
3  days  :  after  a  second  passage  the  mice  died  in  24-48  hours. 


SECTION  II.— MORPHOLOGY  AND  BIOLOGICAL  PROPERTIES. 
1.  Microscopical  appearance. 

The  enterococcus  is  highly  pleomorphic.  In  normal  stools  it  appears  as 
a  diplococcus,  the  cocci  being  rounded,  oval,  or  more  or  less  lancet-shaped, 

very  variable  in  size  and  rarely  encapsulated  ; 
sometimes  the  two  elements  are  arranged  at 
an  angle  to  each  other.  Frequently  the  two 
cocci  in  a  diplococcus  are  of  different  shapes 
and  unequal  in  size,  one  rounded,  the  other 
lancet-shaped. 

In  pus,  in  the  stools  of  cases  of  enteritis, 
and  in  the  blood  of  mice  the  appearance  is 
similar  except  that  many  of  the  cocci  are 
encapsulated  and  look  like  the  pneumococcus. 
Two  diplococci  arranged  in  a  chain  and  also 
— sometimes — diplo-bacilli  may  be  found. 

In  young  cultures  numerous  diplococci, 
tetrads  and  short  chains  are  found  ;  in  older 

FIG.  296. — Enterococcus.     Film  from          ,.  . ,          ,     .  ,  -,   -,  ,-• 

an  agar  culture  exhibiting  the  pieo-    cultures  the  chains  are  longer  and  have  the 
foSifS)? SlekM'  Carbol-blue-    appearance  of  streptococci.     Sometimes,  and 

especially  on  agar,  the  cocci  are  elongated 
and  have  a  bacillary  appearance. 

Staining  reactions. — The  enterococcus  is  easily  stained  by  the  basic  aniline 
dyes.  It  is  gram-positive. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  enterococcus  grows  at  ordinary  temperatures, 
but  best  at  35°-37°  C.  :  it  produces  neither  gas  nor  indol.  Broth  and  agar 
are  very  suitable  media  for  its  growth  ;  liquid  serum  is  not  a  good  medium. 

In  artificial  culture  the  enterococcus  is  indifferently  aerobic.  In  the  tissues 
while  living  as  a  parasite  it  grows  better  under  anaerobic  conditions  and  is 
sometimes  strictly  anaerobic  :  but  this  strict  anaerobiosis  is  merely  temporary 
and  after  passage  through  broth  in  vacuo  the  organism  grows  aerobically 
(Thiercelin,  Rosenthal). 

Culture  media.  Broth. — After  incubating  for  24  hours  the  broth  is  cloudy  ; 
later  it  becomes  clear,  a  whitish  mucous  deposit  falling  to  the  bottom  of  the 
tube. 

Blood-broth. — There  is  a  slight  cloudiness  at  first,  and  then  a  mucous  layer 
containing  numerous  micro-organisms  floats  for  a  time  in  the  liquid,  but 


CULTURAL  CHARACTERISTICS  629 

later  falls  to  the  bottom  of  the  culture  vessel.  Chains  and  capsulated  diplo- 
•cocci  are  found  on  microscopical  examination  of  these  cultures. 

Liquid  serum. — The  growth  in  this  medium  is  very  scanty  and  consists  of 
a  small  glairy  deposit  made  up  of  diplococci  and  encapsulated  cocci  in  chains. 

Agar. — The  growth  consists  of  small  rounded  points  which  are  at  first 
transparent  but  afterwards  opaque  and  often  have  a  streaky  blue  appearance, 
resembling  a  culture  of  streptococci. 

Gelatin. — On  gelatin,  small  white  opaque  rounded  points  are  formed  similar 
to  colonies  of  streptococci :  the  medium  is  not  liquefied. 

Milk. — The  growth  in  milk  is  very  poor.  Coagulation  is  not  a  constant 
phenomenon. 

Potato. — No  apparent  growth  takes  place  on  potato  but  a  microscopical 
examination  of  the  scrapings  of  the  surface  of  the  medium  will  show  that 
there  has  undoubtedly  been  some  multiplication. 

Vitality  and  virulence. — In  liquid  media  the  enterococcus  retains  both  its 
vitality  and  virulence  for  a  long  time  ;  Thiercelin  was  able  to  sow  sub-cultures 
from  a  broth  culture  several  weeks  old,  and  Besson  killed  rabbits  with  a 
culture  which  had  been  sub-cultivated  six  times  in  blood-broth  :  in  anaerobic 
culture  the  vitality  is  maintained  for  several  years.  The  addition  of  appreci- 
able traces  of  antiseptics  (carbolic  acid,  etc,)  does  not  hinder  the  growth  of  the 
cultures. 

Toxins. — The  saprophytic  enterococcus  though  incapable  of  infecting  the  rabbit 
will  nevertheless  often  kill  the  animal  by  toxaemia.  Following  an  injection  of  culture, 
the  animal  sickens  and  becomes  cachectic,  develops  paraplegic  symptoms  and  dies 
on  an  average  in  a  fortnight  to  25  days.  Post  mortem  large  purulent  lesions  are 
frequently  found,  but  the  only  organisms  present  are  organisms  of  secondary  infection 
(staphylococci,  etc.).  Sub-cutaneous  inoculation  of  a  culture  of  the  saprophyte 
filtered  through  a  Chamber-land  bougie  leads  to  the  death  of  the  rabbit  from  cachexia 
(Thiercelin  and  Jouhaud) :  the  same  result  is  obtained  with  cultures  which  have 
been  sterilized  by  boiling  for  half  an  hour  or  by  heating  at  110°  C.  for  15  minutes. 
The  addition  of  iodine  has  no  action  on  the  cachexia-producing  properties  of  the 
toxin  (Rosenthal  and  Charazain). 


SECTION  III.— DETECTION,   ISOLATION  AND  IDENTIFICATION  OP 
THE  ENTEROCOCCUS. 

A.  The  isolation  of  the  enterococcus  from  pathological  material  is  easy  ; 
a,  small  portion  should  be  sown  in  broth  both  aerobically  and  anaerobically. 
and  the  organism  may  then  be  isolated  in  the  ordinary  way  on  sloped  agar 
or  on  plates.     The  colonies  of  the  enterococcus  can  be  recognized  by  their 
characteristic  appearance,  and  may  be  picked  off  and  sown  in  broth  to  obtain 
pure  cultures. 

Note. — In  pathological  material  the  enterococcus,  as  has  already  been 
stated,  often  closely  resembles  the  pneumococcus  and  erroneous  conclusions 
may  be  drawn  from  microscopical  examination  if  this  fact  be  not  borne  in 
mind. 

B.  The  isolation  of  the  enterococcus  from  stools  is  more  difficult  on  account 
of  the  presence  of  other  easily  growing  organisms  :    Thiercelin  describes  two 
methods  : — 

(a)  Dilute  a  trace  of  the  stool  in  a  tube  of  broth,  filter  through  a  double 
layer  of  filter  paper  (the  funnel  and  paper  both  being  sterile)  into  a  sterile 
tube,  and  sow  several  agar-slope  tubes  with  the  filtrate.  After  incubating 
for  24  hours,  the  tubes  will  show  numerous  colonies  of  the  enterococcus  :  the 


630  THE   ENTEROCOCCUS 

majority  of  the  organisms  have  been  retained  on  the  filter,  the  enterococcus 
being  almost  the  only  species  which  has  passed  through. 

(b)  Sow  a  trace  of  the  stool  in  broth,  aspirate  the  liquid  into  a  Roux's 
pipette  and  incubate  at  37°  C.  for  24  hours  in  vacua  ;  then  sow  a  number  of 
agar-slope  tubes  with  the  contents  of  the  pipette  after  diluting  the  latter  in 
sterile  broth.  After  incubating  the  agar  tubes  for  24  hours  at  37°  C.  numerous 
colonies  of  the  enterococcus  are  seen  ;  other  organisms  are  few  in  number. 

C.  The  virulence  of  the  enterococcus  should  be  tested  by  inoculating  mice 
and  rabbits  with  24  hour  old  broth  cultures. 

D.  The  identification  of  the  organism  should  be  based  upon  a  consideration 
of  all  of  the  following  points  : 

1.  The  pleomorphism  of  the  micro-organism  :    in  pathological  exudates 
and  in  mouse's  blood  it  has  the  appearance  of  the  pneumococcus,  in  cultures 
a  few  days  old  it  resembles  the  streptococcus. 

2.  The  growth  on  gelatin  at  20°  C. 

3.  The  minimal  amount  of  growth  in  liquid  serum. 

4.  The  prolonged  vitality  of  the  organism. 

5.  The  fact  that  in  many  cases  it  must  be  grown  anaerobically  before  it 
will  grow  under  aerobic  conditions. 


CHAPTER   XLV. 
MICROCOCCUS  TETRAGENUS. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  631. 

Section  II.— Morphology,  p.  632. 

Section  III. — Biological  properties,  p.  632. 

Section  IV. — Detection,  isolation  and  identification  of  the  organism,  p.  633. 

THE  Micrococcus  tetragenus  was  first  noticed  by  Koch  in  some  pus  from  a 
cavity  in  the  lung.  The  characteristics  of  the  organism  were  studied  by 
Gaffky. 

The  Micrococcus  tetragenus  is  very  commonly  present  on  the  skin  and  is  found 
also  in  the  saliva,  stomach  and  nasal  mucus  of  healthy  persons.  Outside  the  body 
it  occurs  as  a  saprophyte  and  is  often  found  in  the  air.  Under  certain  conditions 
it  may  become  pathogenic :  it  is  frequently  associated  with  the  tubercle  bacillus 
in  tuberculosis  of  the  lung,  in  some  oases  it  is  the  cause  of  sore  throat  ("  angines 
sableuses  "  of  Dieulafoy  and  Appert),  sometimes  of  suppuration  in  very  various 
parts  of  the  body  (purulent  pleurisy,  adenitis,  meningitis,  dental  abscess,  furuncu- 
Josis,  etc.)  and  occasionally  of  septicaemia. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

As  in  the  case  of  the  staphylococci  the  virulence  of  the  Micrococcus  tetragenus 
is  very  variable.  For  purposes  of  experimental  inoculation  a  virulent  strain 
must  be  used. 

Mice. — White  mice  are  very  susceptible.  A  few  drops  of  a  broth  culture 
inoculated  beneath  the  skin  leads  to  death  from  septicaemia  in  24-48  hours ; 
the  blood  and  internal  organs  will  be  found  to  contain  numerous  tetrads. 

Guinea-pigs. — In  guinea-pigs,  sub-cutaneous  inoculation  is  followed  by  the 
formation  of  an  abscess  containing  thick  pus  :  death  may  occur  in  3-5  days, 
but  as  a  rule  the  disease  is  less  acute.  Intra-peritoneal  inoculation  is  a 
more  severe  mode  of  infection  than  the  sub-cutaneous  method  and  leads  to 
a  fatal  result  in  a  few  days :  post  mortem  a  purulent  effusion  will  be  found  in 
the  peritoneal  cavity  and  the  organism  can  be  isolated  from  the  blood  and 
internal  organs. 

Rabbits. — Rabbits  are  less  susceptible.  Sub-cutaneous  inoculation  leads 
to  the  formation  of  a  cold  abscess  (Tissier)  while  intra-peritoneal  inoculation 
is  followed  by  peritonitis  with  a  collection  of  thick  pus  in  the  peritoneal 
cavity. 


632  THE  MICROCOCCUS   TETRAGENUS 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

In  sputum,  pus  and  blood  the  Microccccus  tetragenus  appears  as  single 
cocci,  diplococci  or  as  tetrads.  The  individual  cocci  are  large,  often  exceeding 
1/x  in  diameter ;  they  are  sometimes  oval  and  shaped  like  an  haricot  bean; 
and  are  frequently  surrounded  by  an  irregular  capsule. 

^  In   cultures   on    ordinary  media   the   organism 

'*;•.      '•  grows  as  single  cocci  or  as  diplococci,  the  diameter 

K       •••     seldom  exceeding  0* 6-0' 7/x;  the  tetrad  form  is  rarely 

:v..     ;:$  ''  seen  under  these  conditions,  and  capsules  cannot 

.     .v*    •* "  be  found.     In  cultures  on  liquid  rabbit  serum  the 

''..  ,;      •'•        '   tetrad  forms  with  capsules  can  be  demonstrated. 
*  ':•::•.      ::*         ...  Staining  reactions. — The   micrococcus  is   easily 

:i         \\'fl        stained  by  the  ordinary  methods   and   is   gram- 
•*      ..       .,  positive.     Capsules   may  be  stained  in  the   usual 

way  but  are  often  poorly  defined  and  irregular. 

«  •:• 

FIG.  ^.-Micrococcus  tetm-  *'  Cultural  characteristics. 

f^lX^^S^efch1)111^6'        Tte  Micrococcus  tetragenus  grows  on  the  ordinary 
media  at  temperatures  above   15°  C.  :    at  20°  C. 
growth  is  slow  :   the  optimum  temperature  is  about  37°  C.     It  is  aerobic. 

Culture  media.  Broth. — At  first  the  medium  becomes  slightly  cloudy  but 
as  growth  proceeds  a  thick  ropy  deposit  is  formed. 

Gelatin. — Gelatin  is  not  liquefied.  Single  colonies  grow  as 
small  convex  white  points  1-2  mm.  in  diameter. 

Stab  cultures  give  rise  to  small  isolated  white  colonies  in 
the  depth  of  the  medium  and  a  white  heaped-up  growth  on 
the  surface  (tylotate  growth). 

Chauffard  and  Ramond  have  described  a  strain  of  the  coccus 
which  liquefies  gelatin  to  a  slight  extent.  Chromogenic  varieties 
also  exist,  viz. :  M.  t.  aureus,  M.  t.  subflavus  and  M .  t.  ruber. 

Agar. — On  this  medium  growth  appears  as  white  colonies 
which  become  confluent  and  form  a  creamy  white  viscous 
layer. 

Potato. — Rounded  colonies  appear  which  coalesce  and  form 
a  white  viscous  layer. 

Liquid  rabbit  serum. — The  medium  becomes  cloudy  and  a 
whitish  deposit  consisting  of  capsulated  organisms  is  pre- 
cipitated. 

Milk. — The  organism  grows  feebly  in  milk  and  does  not 
coagulate  the  medium. 


SECTION  HI.— BIOLOGICAL  PROPERTIES. 

Viability.— The  organism  remains  alive  in  culture  media  for 
several  months.     A  temperature  of  60°  C.  for  a  few  minutes    stfb, .  culture 
is  sufficient  to  sterilize  it. 

The  virulence  varies  much  with  different  strains  ;    virulent  strains  retain 
their  properties  in  culture  almost  indefinitely. 

Relying  chiefly  on  differences  in  virulence  some  observers  have  described  several 
varieties  of  the  micrococcus  (M.  t.  septicus,  M.  t.  variaUlis  M.  t.  concentricus,  M.  t. 


THE   MICROCOCCUS   TETRAGENUS  633 

aureus,  etc.)  but  Boldoni  has  shown  that  the  characteristics  of  these  varieties  are 
not  stable.  Pigment  formation  is  not  at  all  constant. 

An  apparently  saprophytic  micrococcus  can  be  made  virulent  by  growing  it  in  a 
collodion  sac  in  the  peritoneal  cavity  of  a  guinea-pig. 

Cultures  sterilized  by  filtration  or  by  heating  at  60°  C.  are  very  feebly 
toxic  and  are  not  pyogenic. 


SECTION  IV.— DETECTION,  ISOLATION  AND  IDENTIFICATION  OF 

THE   ORGANISM. 

On  microscopical  examination  the  organism  will  be  recognized  by  its 
characteristic  appearance.  Isolation  is  easy  on  gelatin  plates — the  medium 
is  not  liquefied.  The  micrococcus  is  differentiated  from  the  staphylococci 
by  the  fact  that  it  does  not  coagulate  milk  [ — some  staphylococci  however 
do  not  clot  milk  (p.  620).] 


FIG.  299. — Micrococcus  tetragenus.     Section  of  a  mouse's  kidney,      x  1200. 

In  those  cases  in  which  the  strain  is  virulent  the  identification  of  the  organism 
may  be  completed  by  the  inoculation  of  a  mouse ;  in  this  animal  the  organism 
will  give  rise  to  a  condition  of  septicaemia  and  the  encapsulated  micrococci 
will  be  found  in  large  numbers  in  the  blood,  internal  organs,  etc. 


CHAPTER   XLVI. 
THE  GONOCOCCUS. 

Introduction. 

Section  I. — Experimental  infection,  p.  635. 

1.  Man,  p.  635.     2.  Animals,  p.  635. 
Section  II. — Morphology,  p.  635. 

1.  Microscopical  appearance  and  staining  reactions,  p.  635.     2.  Cultural  charac- 
teristics, p.  638. 
Section  III. — Biological  properties,  p.  640. 

1.  Vitality  and  virulence,  p.  640.     2.  Toxin,    p.  640.     3.  Serum  therapy.     Agglu- 
tination.    Vaccination,  p.  641. 
Section  IV. — Detection  and  isolation  of  the  gonococcus,  p.  642. 

THE  Gonococcus  is  the  infecting  agent  in  gonorrhoea  (Neisser).  The  organism 
may  be  found  in  cases  of  gonorrhoea  in  the  pus  from  the  urethra  1  and  the 
vagina,  and  in  the  other  lesions  of  the  genital  tract  complicating  gonorrhoea  ; 
for  example,  it  can  be  recovered  from  Bartholin's  glands,  and  from  the 
uterus,  Fallopian  tubes  and  peritoneum  when  these  structures  are  affected. 
The  gonococcus  leads  in  some  cases  to  cystitis,  to  suppurative  affections  of 
the  kidneys,  gonorrhceal  proctitis,  gonorrhceal  ophthalmia  and  the  purulent 
ophthalmia  of  newborn  children. 

It  may  pass  into  the  blood  stream  and  give  rise  to  a  true  septicaemia  (Hallier, 
Krause).  Glon  and  Schlagenhaufer,  and  others  have  recorded  cases  in  which 
the  organism  has  been  found  in  the  lesions  of  gonorrhceal  endocarditis,  and 
Petrone  and  Kammerer  have  found  it  in  the  pus  of  gonorrhceal  arthritis.  As 
a  rule,  it  rapidly  disappears  from  and  cannot  be  found  in  the  exudation 
accompanying  gonorrhceal  rheumatism  because  it  remains  limited  to  the 
articular  tissues  in  which  it  lives  for  a  long  time  but  it  may  be  detected  by 
sowing  cultures  with  the  inflamed  synovial  membrane  (Vaquez).  Bressel 
found  it  in  a  case  of  pneumonia  supervening  on  the  subsidence  of  an  attack 
of  gonorrhoea.  De  Josselin  and  de  Jong  found  the  gonococcus  in  the  cerebro- 
spinal  fluid  of  a  young  man  suffering  from  cerebro -spinal  meningitis  following 
an  attack  of  gonorrhceal  urethritis. 

During  the  first  stage  of  an  attack  of  gonorrhoea  the  gonococcus  is  as  a  rule  present 
in  pure  culture  in  the  urethra.  But  before  long  other  organisms — the  colon  bacillus, 
the  organisms  of  suppuration,  diplococci,  and  various  organisms  described  by  Bumm 

1  Inflammatory  conditions  of  the  urethra  are  sometimes  seen  which  are  not  due  to 
the  gonococcus  (pseudo-gonorrhoea  of  Bockart) ;  among  these  may  be  noted  urethral 
inflammations  due  to  caustics,  to  the  ordinary  organisms  of  suppuration — staphylococci 
and  streptococci  (Bockart),  to  herpes,  to  the  colon  bacillus  (Van  der  Bluyn,  Haay,  Besson), 
to  gout,  rheumatism,  syphilis,  tuberculosis,  etc.  Urethral  inflammations  in  dogs  and 
other  animals  are  caused  by  organisms  other  than  the  gonococcus. 


EXPERIMENTAL  INFECTION  635 

and  others — invade  the  urethra  and  may  either  live  symbiotically  with  the  gonococcus 
or  altogether  displace  the  latter.  These  secondary  organisms  may  be  the  cause  of 
various  complications  of  gonorrhoea,  as  for  instance  abscesses,  suppurations,  endo- 
carditis. 

Though  the  gonococcus  in  many  ways  closely  resembles  the  meningococcus 
and  the  micrococcus  catarrhalis,  these  three  organisms  constitute  distinct 
species. 

SECTION  I.— EXPERIMENTAL  INFECTION. 

Symptoms  and  lesions  in  susceptible  animals.  Man. — (a)  Welander  pro- 
duced gonorrhoea  in  the  human  subject  by  inoculating  pus  containing  the 
gonococcus  into  the  urethra. 

(6)  Bumm  inoculated  a  pure  culture  of  the  gonococcus  into  the  urethra 
of  a  woman  and  set  up  a  typical  attack  of  gonorrhoea  which  lasted  3  weeks  ; 
the  pus  from  the  urethra  contained  the  gonococcus. 

(c)  Bockhart  inoculated  into  the  urethra  of  a  man  in  the  last  stages  of 
general  paralysis  a  gelatin  culture  of  the  gonococcus.     A  typical  attack  of 
gonorrhoea   resulted   which   was   followed   by   suppurative   nephritis ;     the 
gonococcus  was  found  in  pus  from  the  urethra  and  in  the  renal  abscess. 

(d)  Bokai  inoculated  pure  cultures  into  the  urethras  of  six  students  and 
produced  gonorrhoea  in  all  of  them.     Brenner,  Wertheim,  Finger, 'Schlagen- 
haufer,  Keifer  similarly  all  obtained  positive  results. 

Animals. — Animals  are  not  so  susceptible  to  infection  with  the  gonococcus 
as  man.  Sub-cutaneous  inoculation  produces  a  transitory  inflammation  :  in 
one  case  Finger  obtained  a  small  abscess. 

Urethral  inoculations  in  dogs,  rabbits,  horses  and  monkeys  fail  to  produce 
a  clinical  condition  of  gonorrhoea.  Fonseca.  however,  by  inoculating  cultures 
into  the  urethra  of  a  rabbit  produced  a  slight  attack  of  gonorrhoea  which  only 
lasted  a  week  or  10  days. 

Legrain  obtained  a  slight  purulent  conjunctivitis  in  guinea-pigs  ;  gonococci 
were  found  within  the  pus  cells.  In  young  rabbits  the  gonococcus  produces 
a  typical  purulent  conjunctivitis  ;  the  organism  however  does  not  increase 
in  numbers  but  rapidly  disappears  from  the  ocular  conjunctiva.  The  inflam- 
mation is  due  to  the  toxin,  since  sterilized  cultures  give  the  same  result 
(Morax). 

By  inoculating  cultures  into  the  joints  of  rabbits,  Finger  and  Schlagenhaufer 
caused  an  acute  arthritis  which  very  quickly  passed  off. 

By  intra-uterine  inoculation  in  female  rabbits  Malovski  set  up  suppuration 
of  the  Fallopian  tubes  with  peritonitis  which  was  fatal  in  24  hours. 

Inoculation  of  very  virulent  cultures  into  the  peritoneum  of  young  guinea- 
pigs  may  cause  death  from  septicaemia  (Morax).  Pinto  by  inoculating  large 
doses  of  cultures  into  the  peritoneal  cavities  of  very  young  rabbits  produced 
a  septicaemia  with  very  great  difficulty  ;  but  after  passage  through  a  series  of 
rabbits  the  virulence  of  the  organism  was  so  increased  that  a  dose  of  0*000,02  c.c. 
per  kg.  of  animal  weight  was  fatal.  These  researches  have  been  confirmed 
by  Bruckner  and  Christeanu  who  have  succeeded  in  considerably  raising  the 
virulence  of  the  gonococcus  by  passage  through  the  peritoneal  cavities  of 
rabbits  and  cats. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  gonococcus  occurs  as  small  grains  generally  arranged  in  pairs  and 
having  the  appearance  of  two  kidneys  or  haricot  beans  ;  their  long  diameter 


636  THE   GONOCOCCUS 

varies  from  0'6-0'8/x.  The  two  cocci  have  their  concave  faces  adjacent : 
sometimes  they  are  found  grouped  in  staphylococcal  masses  but  never  in 
chains.  In  cultures  the  cocci  are  rounded  or  oval  and  very  unequal  in  size. 
The  two  elements  of  the  diplococcus  are  held  together  by  a  mucous  matrix 
analogous  to  the  capsule  of  the  pneumococcus  but  very  difficult  to  demonstrate. 
It  can  be  stained  in  old  cultures  with  carbol-fuchsin. 


FIG.  300. — Gonococcus  in  pus  from  the  urethra.    Jenner's  stain.    (Oc.  2, 
obj.  ,Mfc,  Zeiss.) 

In  cultures  the  gonococcus  shows  movements  both  of  oscillation  and  of 
translation  (Eraud  and  Hugounenq). 

In  gonqrrhoeal  pus,  the  gonococci  are  sometimes  free  but  more  often  situated 
within  pus  or  epithelial  cells.  This  intra-cellular  position  is  one  of  the 
important  morphological  characteristics  of  the  organism. 


,       '-, 


*-j 


••  «?. 


FIG.  301. — Gonocoecus  in  pus  from  the  urethra.    Pappenheim's  stain.    (Oc.  2, 
obj.  ,'.th,  Zeiss.) 

In  pus  from  the  urethra  the  gonococcus  is  found  in  pure  culture  during  the  first 
few  days.  In  the  early  stages  the  organisms  are  few  in  number  and  are  found 
almost  entirely  within  the  polymorpho-nuclear  leucocytes :  numbers  of  epithelial 


MORPHOLOGY  637 

cells  are  seen  in  the  films  but  very  few  of  them  contain  gonococci.  Towards  the 
third  day,  the  number  of  gonococci  increases  and  a  large  proportion  of  the  leucocytes 
contain  the  organism.  A  little  later  still  the  epithelial  cells  disappear,  and  the 
majority  of  the  gonococci  are  intra- cellular  and  are  so  numerous  that  about  15-20 
per  cent,  of  the  leucocytes  are  invaded.  At  a  later  stage  of  the  disease  secondary 
infections  take  a  part  in  the  inflammatory  process  and  as  the  acute  symptoms  pass 
off  the  epithelial  cells  again  become  numerous  ;  but  it  is  only  when  the  disease 
enters  upon  the  chronic  stage  that  gonococci  are  again  found  within  them  and  the 
pus  cells  diminish  in  number. 

Staining  reactions. — The  gonococcus  stains  easily  with  the  basic  aniline 
dyes  and  is  gram-negative  :  it  is  upon  the  latter  fact  that  its  identification  is 
based  (G.  Roux). 

A.  Stain  first  with  a  single  stain  such  as  dilute  carbol-fuchsin  or  carbol- 
thionin.     All  the  organisms  are  stained. 

B.  Stain  a  film  with  carbol-violet,  examine  under  the  microscope  and  then 
treat  by  Gram's  method.     The  gonococci  will  be  decolourized  and  the  only 
organisms  which  will  retain  the  violet  are  organisms  of  secondary  infection 
such  as  staphylococci,  the  diplococcus  described  by  Legrain,  Bumm,  etc. 

C.  A  film  should  also  be  double  stained  by  one  of  the  methods  based  upon 
the  principle  that  if  a  film  be  stained  by  Gram's  method  the  gram-positive 
organisms  will  be  the  only  organisms  stained,  and  if  at  this  stage  some  other 
dye  in  contrast  to  violet  be  applied  the  gram-negative  organisms  become 
stained  by  it. 

The  following  is  a  list  of  the  special  methods  suitable  for  staining  films 
for  the  detection  of  the  gonococcus  : 

Steinschneider's  method. — 1.  Stain  with  Ehrlich's  violet,  then  with  Gram's 
iodine  solution,  decolourize  in  absolute  alcohol  and  wash  in  water. 

2.  Stain  with  an  aqueous  solution  of  vesuvin,  wash,  dry,  and  mount. 

The  gonococci  and  the  ground  work  are  stained  brown,  and  the  gram- 
positive  organisms  violet. 

Nicolle's  method.  Recommended. — 1.  Stain  with  carbol-violet,  then  with 
Gram's  iodine.  Decolourize  in  acetone-alcohol  (p.  143),  wash  in  water. 

2.  Stain  with  a  drop  of  a  diluted  alcoholic  solution  of  fuchsin  for  a  few 
seconds. 

Saturated  alcoholic  (95  per  cent.)  solution  of  fuchsin,        -  5  c.c. 

Distilled  water,    -  100     „ 

Wash,  dry,  and  mount. 

The  gonococci  are  stained  by  the  fuchsin,  the  other  organisms  by  the  violet. 

Plato's  method. — This  method  depends  upon  the  use  of  neutral  red,  a 
reagent  which  stains  the  living  gonococci  in  fresh  pus  cells,  leaving  the  leuco- 
cytes, the  extra-cellular  gonococci  and  the  secondary  micro-organisms, 
whether  intra-cellular  or  extra-cellular,  unstained. 

The  method  is  useful  for  detecting  gonococci  but  cannot  be  used  for 
permanent  preparations. 

The  staining  solution  should  be  made  up  immediately  before  being  used  : 
Saturated  aqueous  solution  of  neutral-red,       -  1  c.c. 

Normal  saline  solution,  -         •         -         100     „ 

Mix  a  drop  of  pus  with  a  loopful  of  the  stain  on  a  slide,  cover  with  a  cover- 
glass  and  examine  under  the  microscope. 

Wahl's  method. — Wahl  recommends  the  following  stain  for  staining  gono- 
cocci in  sections.  The  solution  keeps  well : — 

Saturated  alcoholic  solution  of  auramine,         -  2      c.c. 

95  per  cent,  alcohol,     -          -  1*5     ,, 

Saturated  alcoholic  solution  of  thionin,  -  2        „ 

Saturated  aqueous  solution  of  methyl  green,  -  3        ,, 

Distilled  water, 6 


638  THE   GONOCOCCUS 

[Jenner's  stain  and  Pappenheim's  stain  are  both  useful  for  staining  films 
of  pus  when  searching  for  the  gonococcus.] 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  gonococcus  is  an  aerobic  organism  which  can 
only  be  cultivated  outside  the  body  with  some  difficulty.  It  is  necessary 
to  use  special  media  and  those  containing  serum  give  the  best  results.  The 
optimum  temperature  is  36°-37°  C.  though  growth  takes  place  at  all  tem- 
peratures between  21°  and  39°  C.  The  gonococcus  ferments  glucose  but  no 
other  sugar. 

Culture  media.  Broth. — In  ordinary  broth  at  a  temperature  of  36°  or  37°  C. 
the  amount  of  growth  is  insignificant :  towards  the  second  day  the  medium 
is  cloudy,  but  subsequently  becomes  clear,  a  slight  greyish  deposit  falling  to 
the  bottom  of  the  vessel.  Vanned  recommends  veal  broth  containing  no 
peptone  as  a  useful  culture  medium.  The  broth  is  evaporated  to  one-fourth 
its  volume  and  then  one-third  its  volume  of  ascitic  fluid  is  added. 

Bruschettini  and  Ansaldo  obtained  abundant  growths  by  adding  a  few 
drops  of  egg-yolk  or  egg-albumin  to  ordinary  broth.  According  to  these 
observers  the  best  medium  is  :— 

Sterile  beef  broth,  10  c.c. 

Defibrinated  blood,       ......  1  drop. 

Fresh  white  of  egg,       -  1       „ 

Urine  (Finger). — In  non-alkaline  urine  to  which  O5  per  cent,  peptone  has 
been  added  and  the  whole  sterilized,  the  gonococcus  grows  better  than  in 
broth.  It  produces  a  well-marked  turbidity  and  a  fairly  copious  precipitate. 
Hammer  prefers  a  highly  albuminous  urine  sterilized  either  in  the  autoclave 
or  by  filtration  through  porcelain. 

Acid  gelatin  (Turro). — The  gonococcus  grows  quite  well  at  22°  C.  in  an  acid 
gelatin — ordinary  gelatin  which  has  not  been  neutralized — but  the  cultures 
are  delicate  and  growth  fails  after  the  third  or  fourth  sub-culture.  The 
medium  is  not  liquefied. 

Stab  culture. — After  incubating  for  several  days  a  very  delicate  white  line 
appears  along  the  line  of  the  stab .  The  amount  of  growth  is  always  very  poor. 

Single  colonies, — Single  colonies  appear  as  raised,  viscous,  white,  hemi- 
spherical points  which  always  remain  very  small  in  size. 

Ordinary  agar. — A  very  scanty  growth  is  obtained  by  sowing  gonorrhceal 
pus  on  the  surface  of  ordinary  agar  :  the  necessary  albuminoid  material  is 
provided  by  the  pus.  The  culture  takes  the  form  of  a  very  thin  glaze. 

Cultures  have  very  little  viability.  It  seems  impossible  to  sub-cultivate  on  agar 
for  more  than  a  generation  or  two  and  growth  ceases  generally  after  the  second 
sub-culture.  Wildbolz  however  by  using  a  slightly  alkaline  agar  has  occasionally 
succeeded  in  sub-cultivating  for  several  generations.  Thalmann  recommends 
ordinary  agar  made  neutral  to  phenolphthalem.  Vanned  uses  ordinary  1*5  per 
cent,  agar  made  alkaline  with  10  per  cent,  solution  of  sodium  carbonate  so  that  the 
mixture  turns  litmus  paper  very  slightly  blue. 

Wertheim's  agar. — Tubes  of  sterilized  agar  (about  6  c.c.  in  each  tube)  are 
liquefied  by  heat  and  cooled  to  45°  C.  To  each  tube  about  4  c.c.  of  human 
blood  serum  or  sterile  ascitic  fluid  are  added  with  aseptic  precautions.  The 
contents  of  the  tubes  are  mixed,  sloped  and  allowed  to  cool.  The  tubes  are 
sown  when  the  medium  has  set. 

It  is  an  advantage  both  in  this  and  in  the  following  media  to  use  glycerin-agar 
instead  of  ordinary  agar. 

Stroke  cultures. — Stroke  cultures  incubated  for  2-3  days  at  37°  C.  give  a 
narrow,  thin,  greyish,  semi  transparent  band  with  a  moist  lustrous  surface. 


CULTURAL  CHARACTERISTICS 


639 


Single  colonies. — To  obtain  single  colonies  Wertheim's  agar  may  be  poured 
into  Petri  dishes  and  after  solidifying  sown  by  the  parallel  stroke  method 
(p.  81).  After  incubating  for  24  hours  at  37°  C.  small,  punctiform,  viscous 
and  transparent  colonies  appear  ;  these  increase  in  size  and  about  the  second 
or  fourth  day  may  be  as  large  as  a  pin's  head  and  hemispherical ;  examined 
with  a  lens,  their  edges  are  seen  to  be  slightly  sinuous  and  their  centres  whitish, 
semi-opaque  or  even  opaque  (Wildbolz). 

Krai's  agar. — Krai  substitutes  calf  for  human  serum  in  Wertheim's  agar. 

Heiman's  agar. — Proceeding  as  above,  mix  two  parts  of  ordinary  agar  and  one 
part  of  pleural  fluid  sterilized  by  discontinuous  heating  (p  47).  The  medium 
should  be  neutral  and  if  the  serum  be  alkaline  it  should  be  added  to  a  slightly  acid 
agar. 

Wildbolz'  agar. — Heffter  having  shown  that  fluid  from  ovarian  cysts — which 
contains  a  large  amount  of  pseudo-mucin — may  with  advantage  be  used  instead  of 
ascitic  fluid  in  the  preparation  of  media  for  the  gonococcus,  Wildbolz  recommends 
an  agar  containing  pseudo-mucin. 

Melt  some  ordinary  agar  and  add  5  per  cent,  of  finely  powdered  pseudo-mucin.1 
Heat  the  mixture  to  100°  C.  for  an  hour,  filter  while  hot,  distribute  into  tubes  and 
sterilize  at  100°  C. 

Abe's  agar. — Take  500  grams  of  beef,  cut  off  all  the  fat,  mince  and  macerate 
in  1  litre  of  water  for  20  hours  in  the  ice  chest.     Filter 
through  paper,  then  through  a  Chamberland  bougie. 

To  the  nitrate  add  ordinary  nutrient  agar  which  has 
been  melted  and  cooled  to  50°  C.  in  the  proportion  of  two 
parts  of  meat  extract  to  five  of  agar.  Distribute  in  tubes 
or  Petri  dishes.  The  medium  is  transparent  and  very  suit- 
able for  the  growth  of  the  gonococcus. 

Pfeiffer's  blood-agar. — This  medium  is  prepared  by 
simply  spreading  a  few  drops  of  sterile  fresh  human  blood 
on  agar  plates.  The  characteristics  of  the  growth  are  the 
same  as  those  on  Wertheim's  agar  (Ghon  and  Schlagen- 
haufer,  Abel). 

Bezanpon  and  Griffon's  blood-agar  (p.  53). — This  is  a 
very  useful  medium  for  growing  the  gonococcus. 

Tubes  of  blood-agar  sown  with  a  liberal  amount  of 
gonorrhceal  pus  and  incubated  at  37°  C.  show,  after  24 
hours,  a  copious  growth  consisting  of  flat,  rounded,  moist, 
transparent,  lustrous  colonies  of  variable  size,  sometimes 
coalescing  to  form  a  viscous  layer  with  pinked  edges.  The 
medium  which  is  in  the  first  instance  red  becomes  chocolate- 
coloured  as  growth  proceeds,  the  colonies  being  delicately 
picked  out  in  white. 

Nasstikoff's  agar. — This  medium  is  recommended  by 
Steinschneider  for  the  cultivation  of  the  gonococcus .  Collect 
the  yolk  of  an  egg  in  a  sterile  manner  (p.  54)  and  mix  it 
thoroughly  with  three  times  its  volume  of  sterile  water. 
Liquefy  a  few  tubes  of  sterile  agar  each  containing  about 
6  c.c.,  and  when  they  have  cooled  to  45°  C.  add  2  c.c.  of  the  egg  emulsion 
to  the  contents  of  each  tube,  being  careful  to  avoid  contaminations.  Mix 
carefully,  slope  the  tubes  and  allow  to  set. 

Leipschutz"  agar. — Prepare  a  2  per  cent,  solution  of  the  substance  sold  by 
Merck  as  "  powdered  egg-albumin."  To  100  c.c.  of  this  solution  add  20  c.c. 
of  deci-normal  soda  solution.  Stand  for  half  an  hour  and  filter  through 


FIG.  302.— Gonococ- 
cus. Stroke  culture  on 
glycerin-ascitic-agar  (3 
days  at  37°  C.). 


1  Pseudo-mucin  is  obtained  by  precipitating  the  fluid  from  ovarian  cysts  with  alcohol. 


640  THE   GONOCOCCUS 

filter  paper.  Distribute  in  small  Erlenmeyer  flasks  and  sterilize  by  heating 
at  100°  C.  on  three  separate  occasions  during  the  next  24  hours.  Then  add 
the  liquid — which  is  colourless  or  pale  yellow,  transparent,  and  alkaline  to 
litmus — to  ordinary  nutrient  agar  in  the  proportion  of  1-3  of  agar.  A  broth 
may  be  prepared  with  albumin  in  a  similar  manner. 

Milk-agar. — Milk  added  to  glycerin-agar  constitutes  a  very  good  medium 
for  the  growth  of  the  gonococcus  (Bruschettini  and  Ansaldo). 

Steinschneider's  agar  is  prepared  by  mixing  one  part  of  human  urine 
collected  aseptically  with  two  parts  of  sterile  agar  (the  technique  is  the  same 
as  for  Wertheim's  agar). 

Bumm's  serum. — Bumm  uses  solidified  human  serum.  The  blood  is 
collected  with  aseptic  precautions  during  confinement. 

Immediately  after  cutting  the  umbilical  cord,  the  placenta  being  still  in  the 
uterus,  wash  the  central  end  in  corrosive  sublimate  solution,  and  allow  the  blood 
to  flow  into  a  sterile  flask.  In  this  way  as  much  as  100  c.c.  of  blood  can  be  collected 
and  from  the  blood  a  perfectly  clear  serum  can  be  drawn  off  18-24  hours  later  which 
is  then  solidified  in  the  ordinary  way. 

It  is  easier  to  use  blood  collected  from  a  vein  of  the  forearm  (p.  193).  The 
characteristics  of  the  growth  on  human  serum  are  the  same  as  on  Wertheim's 
agar. 

De  Christmas  uses  coagulated  rabbit  serum  instead  of  human  serum  and 
cultivates  the  organism  in  tubes  of  small  diameter. 

Potato. — No  growth  takes  place  on  potato.  On  glycerin-potato  Bruschettini 
and  Ansaldo  obtained  a  good  growth  by  sowing  from  a  white-of-egg-broth 
culture. 

SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.  Viability.    Virulence. 

Gonorrhceal  pus  is  sterilized  in  a  few  minutes  by  heating  at  a  temperature 
of  55°  C.  Similarly  if  kept  at  a  temperature  of  0°  C.  in  an  ice  chest  for  a 
few  hours  cultures  cannot  be  obtained  on  sowing  the  material.  Desiccation 
and  exposure  to  the  air  will  rapidly  sterilize  gonococci  in  pus.  The  weakest 
antiseptics  are  sufficient  to  kill  the  organism. 

The  gonococcus  has  very  little  viability  in  cultures.  If  kept  in  the  incubator 
the  cultures  may  live  2  or  3  weeks  but  if  kept  at  the  ordinary  temperature  only 
a  few  hours.  Successive  sub-cultures  soon  become  unfertile  ;  as  a  rule  sub- 
cultures fail  to  grow  after  the  fourth  or  fifth  generation,  but  on  media  con- 
taining albumin,  Leipschutz  succeeded  in  cultivating  the  organism  for  thirty- 
five  generations. 

Young  cultures  on  suitable  media  are  virulent  but  the  virulence  is  soon 
lost.  The  lesions  produced  in  animals  by  cultures  are  chiefly  due  to  the 
toxin  inoculated  at  the  same  time.  The  virulence  of  any  strain  of  the  organism 
may  be  increased  by  passage  through  rabbits  (p.  635). 

2.  Toxin. 

The  toxin  of  the  gonococcus  was  studied  by  De  Christmas,  and  his  results 
were  confirmed  by  Wassermann  and  Nicolaysen.  These  observers  found 
that  the  toxin  is  almost  exclusively  intra-cellular  whence  it  diffuses  only 
very  slowly  into  the  surrounding  media.  Morax  and  Elmassian  obtained 
an  active  toxin  by  macerating  gonococci  in  a  1  per  cent,  solution  of  potash 
for  a  week  or  10  days. 

Preparation  of  the  toxin. — For  the  cultivation  of  the  organism  De  Christmas 
at  first  used  a  mixture  of  one-third  ascitic  fluid  and  two-thirds  peptone 


SERUM  THERAPY  641 

broth  ;  he  now  prefers  a  mixture  of  meat  extract  and  ascitic  fluid.  After 
incubation  the  growth  is  filtered. 

Macerate  500  grams  of  fresh  minced  veal  for  a  few  hours  in  a  litre  of  warm  water  : 
add  2  or  3  grams  of  gelatin  at  will,  but  no  salt.  Heat  to  105°  C.  for  half  an  hour. 
Filter,  concentrate  to  one-fourth  its  original  volume  and  sterilize.  To  25  parts  of 
this  mixture  add  75  parts  of  ascitic  fluid. 

The  gonococcus  must  be  acclimatized  to  the  medium  by  growing  successively  in 
broth  containing  one-fourth  then  one- half  of  its  volume  of  ascitic  fluid.  After 
acclimatization  the  resistance  and  the  toxigenic  capacity  of  the  organism  increase. 
The  medium  should  be  sown  with  a  young — 2  or  3  days- old — culture  on  rabbit  serum  ; 
the  organism  will  remain  alive  in  the  liquid  for  6  weeks. 

Twelve  hours  after  sowing  the  medium  is  cloudy  and  the  surface  is  covered  with 
a  creamy  pellicle ;  later  the  fluid  becomes  clear,  the  growth  being  deposited  as  a 
greyish  viscous  precipitate  throwing  out  prolongations  which  float. 

The  toxin  content  is  at  its  maximum  after  the  culture  has  been  incubating  for 
3-4  weeks  at  37°  C. 

Properties. — The  toxin  has  all  the  properties  of  the  diastases.  It  is  pre- 
cipitated from  filtered  cultures  by  strong  alcohol  and  sulphate  of  ammonium. 
It  is  soluble  in  glycerin.  It  can  be  heated  to  65°  C.  for  15  minutes  without 
undergoing  any  change,  but  between  65°  and  75°  C.  its  properties  are  altered 
and  at  75°-80°  C.  it  is  soon  destroyed.  It  does  not  dialyse  through  parchment. 

The  gonotoxin  is  very  toxic  to  laboratory  animals  though  they  are  immune 
to  the  gonococcus  itself. 

Sub- cutaneous  or  intra-peritoneal  inoculation  of  1—2  c.c.  of  the  toxin  will  some- 
times kill  guinea-pigs  though  generally  it  is  necessary  to  inoculate  5—10  c.c.  of  toxin 
intra-peritoneally  to  produce  a  fatal  result  in  these  animals.  Sub-cutaneous  inocula- 
tion in  guinea-pigs  and  rabbits  is  followed  by  the  formation  of  an  abscess  which 
soon  becomes  the  seat  of  secondary  infections.  The  injection  of  toxin  into  the 
pleural  cavity  of  a  rabbit  produces  a  purulent  but  sterile  effusion. 

Gonotoxin  injected  intra-cerebrally  in  doses  of  0*002  c.c.  kills  guinea-pigs  in  4-6 
hours.  Smaller  doses  are  not  fatal  but  lead  to  a  high  degree  of  immunity  so  that 
the  animal  can  resist  further  in tra- cerebral  inoculations.  Large  doses  of  toxin 
inoculated  sub-cutaneously  on  several  different  occasions  produce  an  immunity 
against  intra- cerebral  inoculation. 

In  man,  a  distinct  urethritis  is  produced  by  injecting  2  c.c.  of  a  1  in  10 
dilution  of  toxin  into  the  fore  part  of  the  urethra  and  leaving  it  there  for 
2  or  3  minutes. 

3.  Serum  therapy.    Agglutination.    Vaccination. 

Goats  which  have  been  treated  with  large  doses  of  gonotoxin  inoculated 
into  the  sub-cutaneous  cellular  tissue  yield  an  antitoxic  serum  (De  Christmas). 

A  mixture  of  toxin  and  antitoxic  serum  is  harmless.  Neutralization  does  not 
occur  immediately  but  takes  3-4  hours  :  at  15°  C.  one-half  of  a  cubic  centimetre 
of  the  serum  neutralizes  10  c.c.  of  the  toxin. 

If  injected  alone  into  the  brain  before  the  toxin,  the  serum  protects  the  animal 
for  3  days  against  an  intra- cerebral  inoculation  of  toxin,  but  if  inoculated  after 
the  toxin  it  has  neither  prophylactic  nor  therapeutic  properties. 

Injected  sub-cutaneously  or  into  the  veins  or  peritoneal  cavity  in  large  doses 
(1-5  c.c.)  the  serum  has  prophylactic  properties  provided  that  the  inoculation  of 
toxin  be  not  made  until  after  an  interval  of  48  hours. 

Vanned  also  prepared  a  rabbit  serum  which  had  therapeutic  properties 
against  the  gonococcus  toxin  and  agglutinated  the  organism  in  dilutions  of 
1  in  200  to  1  in  400. 

Rogers  and  Torrey  immunized  sheep  by  intra-peritoneal  inoculation. 
Cultures  heated  to  65°  C.  for  half  an  hour  were  used  for  the  first  inoculations, 
then  living  cultures.  The  serum  agglutinated  the  organism  and  according 

2s 


642  THE   GONOCOCCUS 

to  these  observers  was  of  therapeutic  value  in  gonorrhoeal  rheumatism  and 
gonorrhoeal  infections  of  the  geni to-urinary  tract  in  man. 

The  serum  of  guinea-pigs  and  rabbits  immunized  by  intra-peritoneal 
inoculations  of  cultures  of  the  gonococcus  agglutinates  old  cultures  of  the 
gonococcus  and  also,  but  to  a  less  extent,  young  cultures. 

Bruckner  and  Christeanu  after  inoculating  a  horse  on  several  occasions  with 
cultures  of  the  gonococcus  obtained  a  powerfully  agglutinating  serum.  This 
serum  exhibited  therapeutic  properties  against  an  intra-peritoneal  inoculation 
in  rabbits. 

The  serum  of  a  person  suffering  from  gonorrhoeal  prostatitis  agglutinated 
an  old  culture  but  not  a  young  culture  (Wildbolz). 

Normal  human  serum  and  normal  guinea-pig  serum  did  not  possess  agglu- 
tinating properties. 

[Human  vaccine  therapy. — Vaccines  prepared  by  Wright's  method  (p.  605) 
give  distinctly  encouraging  results  in  the  treatment  of  gonorrhoea  and  its 
complications.  An  autogenous  vaccine  must,  of  course,  be  used  to  obtain 
the  most  satisfactory  results  but  while  this  is  in  preparation  a  "  stock  " 
vaccine — prepared  from  a  laboratory  strain — may  be  administered  to  avoid 
delay  in  the  treatment.  The  initial  dose  should  consist  of  five  or  ten  million 
organisms  and  the  amount  inoculated  on  subsequent  occasions  must  be 
determined  by  the  extent  of  the  local  inflammation  and  by  the  degree  of 
general  malaise  following  each  dose.  It  is  important  that  a  severe  reaction 
be  not  excited.] 

SECTION  IV.-DETECTION  AND  ISOLATION  OF  THE   GONOCOCCUS. 

Pus  from  the  urethra  and  from  the  various  secondary  suppurations  should 
form  the  material  for  examination. 

In  collecting  pus  from  the  urethra,  after  cleansing  the  meatus  squeeze 
the  penis  from  the  root  to  the  glans,  and  aspirate  the  pus  into  a  pipette  or 
collect  it  on  a  platinum  loop. 


'  »    -*  n  4 


*i 

FIG.  303.— Gonococcus  in  pus  from  the  urethra.     Gram's  stain  and  dilute 
carbol-fuchsin.     (Oc.  2,  obj.  Tl2th,  Zeiss.) 

(a)  Microscopical  examination. — In  making  film  preparations  the  cover- 
glasses  should  not  be  pressed  together  too  firmly  and  should,  be  slid  apart 
gently  so  that  the  pus  cells  are  not  broken  :  if  the  films  be  treated  roughly 


ISOLATION  OF  THE   ORGANISM  643 

the  gonococci  will  be  set  free,  and  one  of  the  characteristic  appearances  lost. 
The  film  should  be  stained  by  methods  which  will  differentiate  the  gonococcus 
from  other  organisms  likely  to  be  found  with  it. 

In  medico-legal  cases  it  sometimes  happens  that  pus  dried  on  linen  has  to  be 
examined  for  the  gonococcus.  Heger-Gilbert  recommends  that  in  such  circumstances 
the  material  should  be  dealt  with  as  follows : 

Cut  out  the  piece  of  linen  on  which  the  pus  has  dried  and  lay  it  in  a  watch-glass 
on  a  piece  of  filter  paper  which  has  been  thoroughly  saturated  with  slightly  alkaline 
normal  saline  solution.  Cover  with  another  watch-glass  and  leave  for  1—5  hours. 
Then  apply  the  fine  end  of  a  pipette  to  the  different  parts  of  the  linen  and  aspirate 
gently.  The  formed  elements  in  the  pus  are  thus  sucked  up  with  a  little  fluid  into 
the  pipette.  Spread  the  fluid  collected  in  the  pipette  on  a  slide,  dry  and  stain  as 
already  directed  (vide  ante). 

(b)  Cultures. — To  isolate  the  gonococcus  in  culture  the  pus  should  be  col- 
lected during  the  first  few  days  of  the  disease  ;  later,  infection  of  the  urethra 
with  other  organisms  will  complicate  the  technique.  It  is  best  to  sow  stroke 
cultures  on  plates  of  serum-agar  or  blood-agar  and  to  aim  at  getting  isolated 
colonies. 

Note. — In  chronic  gonorrhoea,  when  the  discharge  is  reduced  to  a  mere  drop,  the 
best  method  of  finding  the  gonococcus  is  to  instruct  the  patient  to  pass  his  urine 
on  waking  into  a  conical  vessel,  and  to  let  it  stand,  adding  a  crystal  of  thymol  as 
an  antiseptic.  In  a  short  time  whitish  threads  of  mucus  or  muco-pus  are  deposited 
at  the  bottom  of  the  vessel.  Aspirate  the  deposit  with  a  pipette  and  prepare  films 
as  already  described.  This  method  cannot  be  applied  ,to  the  isolation  of  the  gono- 
coccus, and  if  it  be  necessary  to  obtain  cultures  the  urine  must  be  collected  in  a 
sterile  manner  in  a  sterile  vessel,  and  a  thread  at  once  removed  with  a  sterile  pipette 
and  sown  on  serum-agar.  It  is  sometimes  useful  to  centrifuge  the  urine  and  examine 
the  deposit  for  the  gonococcus. 

In  cases  of  gonorrhoea  of  old  standing  when  the  pus  contains  very  few  gonococci 
it  often  happens  that  the  latter  cannot  be  found.  Neisser  has  shown  that  a  local 
or  general  stimulant,  e.g.  an  injection  of  nitrate  of  silver  locally  or  the  consumption 
of  beer,  leads  in  such  cases  to  an  increase  in  the  amount  of  pus,  and  in  this  it  may 
be  possible  to  detect  the  gonococcus. 


CHAPTER  XLVII. 
THE  MENINGOCOCCUS. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  645. 

Section  II. — Morphology,  p.  645. 

1.  Microscopical  appearance  and  staining  reactions,  p.  645.     2.  Cultural  charac- 
teristics, p.  647. 
Section  III. — Biological  properties,  p.  647. 

1.  Bio-chemical  reactions,  p.  647.     2.  Vitality  and  virulence,  p.  648.     3.  Toxins, 
p.  648.     4.  Immunization  and  serum  therapy,  p.  648.     5.  Agglutination,  p.  649. 
Section  IV. — The  isolation  and  identification  of  the  meningococcus,  p.  650. 

1.  The  diagnosis  of  meningococcal  meningitis,  p.  650.     2.  The  isolation  of  the 
organism,  p.  650. 

Micrococcus  catarrhalis,  p.  651. 

EPIDEMIC  cerebro-spinal  meningitis  x  is  generally  caused  by  the  Diplococcus 
intra-cellularis  meningitidis  discovered  by  Weichselbaum  and  now  generally 
known  as  the  Meningococcus. 

Though  the  Meningococcus  is  the  most  frequent  cause  of  the  disease  (especially 
in  recent  epidemics)  it  is  of  course  not  the  only  cause  of  epidemic  meningitis. 

In  some  cases  of  epidemic  meningitis  a  typical  pneumococcus  has  been 
recovered  from  the  cerebro-spinal  fluid  (Won0,  Netter  and  others),  and  Marchoux 
described  an  epidemic  of  cerebro-spinal  meningitis  in  Senegal  which  was  due  to  the 
pneumococcus. 

In  another  epidemic  of  cerebro-spinal  meningitis  Jaeger  and  Heubner  found  an 
organism  quite  distinct  from  the  Meningococcus  (p.  626).  Bonome,  again,  described 
an  encapsulated  gram-positive  streptococcus  as  the  cause  of  an  epidemic  of  cerebro- 
spinal  meningitis  :  this  organism  has  also  been  demonstrated  in  a  large  number 
of  cases  of  meningitis,  particularly  by  Netter :  it  is  commonly  found  in  association 
with  the  Meningococcus. 

The  Meningococcus  is  present  in  the  cerebro-spinal  fluid  and  in  the  fluid 
exudate  on  the  meninges  in  cases  of  meningitis  :  it  occasionally  enters  the 
blood  stream  (Salomon,  Elser,  Sacquepee,  etc.).  It  has  been  shown  that 
meningococcal  cerebro-spinal  meningitis  is  always  preceded  by  a  more  or  less 
well  marked  naso-pharyngitis,  with  symptoms  of  sore  throat  and  coryza,  due 
to  infection  with  the  meningococcus  (Strumpell,  Weigert,  Albrecht  and  Ghon, 
Kiefer,  Jundell,  etc.).  According  to  Ostermann  it  is  this  naso-pharyngitis  which 
is  contagious  and  disseminates  the  meningococcus.  Some  individuals  affected 
with  pharyngitis  escape  an  attack  of  meningitis,  which  only  supervenes  when 
the  organism— under  conditions  still  little  understood—passes  from  the  naso- 

[*The  expression  "epidemic  meningitis"  is  very  commonly  used  to  connote  meningo- 
coccai  meningitis.  Such  a  limitation  of  the  meaning  is,  of  course,  very  misleading.] 


EXPERIMENTAL  INOCULATION  645 

pharynx  to  the  meninges.  During  an  epidemic  of  meningococcal  cerebro-spinal 
meningitis  it  is  therefore  a  matter  of  great  importance  that  the  naso-pharynx 
of  contacts  should  be  examined  for  the  presence  of  the  Meningococcus :  a 
person  may  carry  the  organism  in  his  naso-pharynx  and  spread  the  infection 
while  himself  remaining  in  apparently  good  health.  Indeed  Bruns  and  Hohn 
venture  the  opinion  that  there  are  ten  to  twenty  times  more  carriers  of  the 
Meningococcus  than  cases  of  meningitis.  On  an  average  the  Meningococcus 
remains  in  the  pharynx  of  a  "  carrier  "  for  about  a  fortnight,  though  it  may 
persist  for  several  months. 

Meningococcus  and  Gonococcus. — Many  observers  have  drawn  attention 
to  the  close  resemblance  which  exists  between  the  Meningococcus  and  the 
Gonococcus,  e.g.  their  microscopical  appearances,  reaction  to  Gram's  stain, 
intra-cellular  position  within  the  leucocytes  and  agglutination  by  the  same 
specific  serums. 

It  has  however  been  shown  that  these  two  organisms  though  they  belong 
to  the  same  group  are  nevertheless  two  sharply  differentiated  species  :  the 
Meningococcus  is  pathogenic  for  mice  while  the  Gonococcus  is  not,  and  the 
cultural  characteristics  of  the  two  cocci  are  different :  Zupnik  by  inoculating 
cultures  of  the  second  generation  of  the  Meningococcus  into  the  urethra  of 
five  medical  men  in  good  health  failed  in  every  case  to  produce  symptoms  of 
gonorrhoea,  while  if  a  gonococcus  even  of  the  twentieth  generation  be  simi- 
larly inoculated  symptoms  of  gonorrhoea  follow.  Finally  the  two  organisms 
can  be  shown  to  be  different  species  by  a  study  of  the  agglutination  and 
complement  fixation  reactions. 

SECTION  I.— EXPERIMENTAL  INOCULATION. 

The  virulence  of  the  Meningococcus  is  subject  to  considerable  variation 
but  in  any  case  it  is  only  slightly  pathogenic  for  laboratory  animals 

Mice  are  the  most  susceptible  animals  :  they  generally  succumb  after  intra- 
peritoneal  inoculation  of  a  large  dose  of  culture.  Post  mortem  examination 
reveals  a  condition  of  peritonitis,  and  in  the  exudate  the  micro-organisms  will 
be  found  to  be  present  in  very  large  numbers  ;  the  spleen  and  the  heart  blood 
contain  very  few. 

Rabbits  and  especially  guinea-pigs  are  less  susceptible  :  they  only  suc- 
cumb after  the  inoculation,  intra-peritoneally  or  intra-venously,  of  very 
large  doses  of  cultures.  In  these  animals  metastatic  deposits  do  not  occur 
and  the  organism  does  not  multiply ;  death  results  from  intoxication 
(Weichselbaum,  Jundell).  In  mice  on  the  other  hand  some  slight  degree  of 
multiplication  seems  to  take  place  but  successive  passages  never  lead  to  any 
increase  in  virulence  (Bettencourt  and  Franca). 

Inoculation  into  the  meninges  of  laboratory  animals  does  not  lead  to 
symptoms  of  meningitis  (Weichselbaum,  Albrecht  and  Ghon).  But  in 
monkeys,  Flexner  produced  a  true  meningitis  fatal  in  24  hours  by  inoculating 
the  Meningococcus  beneath  the  arachnoid  :  post  mortem,  the  lesions  typical  of 
cerebro-spinal  meningitis  were  found. 

SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

In  meningeal  exudates  and  in  the  cerebro-spinal  fluid  the  Meningococcus 
generally  occurs  as  a  diplococcus.  The  elements  composing  the  diplococcus 
resemble  coffee  beans,  the  flattened  surfaces  being  opposed  to  each  other. 
The  micro-organism  is  very  similar  to  the  Gonococcus  in  appearance  :  not 


646  THE  MENINGOCOCCUS 

infrequently  two  diplococci  are  seen  arranged  as  tetrads.  Isolated  individuals 
are  round  and  of  variable  size  :  chains  are  never  seen.  Most  of  the  organisms 
are  contained  within  the  leucocytes,  some  of  the  latter  being  crowded  with 
cocci. 


FIG.  304.  —  Meningococcal  exudate.    Jenner's  stain.    (Oc.  2,  obj.  ^th.     Zeiss. 

In  the  cerebro-spinal  fluid  and  in  meningeal  exudates  the  Meningococcus 
is  rarely  encapsulated,  but  exceptionally  individuals  show  a  very  distinct 
membrane.  In  cultures  on  rabbit  serum  the  capsules  are  very  apparent. 

In  cultures,  as  in  pathological  exudates,  the  cocci  may  occur  singly,  or  in 
pairs,  or  as  very  distinct  tetrads  :  they  are  frequently  found  in  small  agglu- 
tinated clumps  :  chains  have  never  been  observed.1 

Staining  reactions.  —  The  Meningococcus  is  readily  stained  by  the  basic 
aniline  dyes  ;  carbol-blue  and  carbol-thionin  are  especially  useful. 

The  organism  is  gram-negative. 


ft 


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fi 

FIG.  305.  —  Meningococcai  exudate.    Gram's  stain  and  eosin.    (Oc.  2,  obj.  T^th.     Zeiss.) 

Schottmiiller  examined  43  strains  and  found  that  they  were  all  decolourized  by 
Gram's  method  even  after  being  sub-cultivated  many  times.     But  according  to 

1  The  Jaeger-  Heubner  diplococcus,  on  the  other  hand,  forms  long  chains  in  liquid  media. 


MORPHOLOGY  647 

Lehmann  it  is  not  uncommon  to  find  gram-positive  individuals  in  preparations  of 
the  Meningococcus. 

2.  Cultural  characteristics. 

Conditions  of  growth. — The  Meningococcus  is  a  strict  ae'robe.  Growth 
will  only  take  place  at  temperatures  between  25°  C.  and  42°  C.,  the  optimum 
temperature  being  37°  C.  In  attempting  to  isolate  the  organism  it  maybe 
difficult  at  first  to  obtain  cultures,  but  once  established  growth  takes  place 
quite  well  on  the  ordinary  media.  Media  which  contain  blood  or  serum 
(such  as  human-blood-agar,  ascitic-agar  and  liquid  rabbit  serum)  are  particu- 
larly useful,  and  should  be  employed  for  the  primary  cultures. 

Characters  of  growth.  Agar. — When  large  quantities  of  pus  rich  in  meningo- 
cocci  are  sown  on  agar  a  small  number  of  colonies  appear.  In  24  hours  these 
attain  a  diameter  of  2  mm.,  are  raised — with  the  surface  flattened  and  borders 
rounded — greyish,  and  at  first  transparent  subsequently  becoming  opaque 
in  the  centre. 

Blood-agar.  Ascitic-agar. — Early  sub-cultures  give  isolated  colonies  having 
the  same  characteristics  as  those  on  agar,  but  after  several  sub-cultures  the 
colonies  become  confluent  and  form  a  copious  greyish  deposit  with  wavy 
edges. 

Kutscher  recommends  agar  made  with  placenta.  Macerate  a  chopped-up  placenta 
in  twice  its  weight  of  water,  and  to  each  100  parts  of  maceration  add  2*5 
parts  agar,  0'5  parts  salt,  1  part  glucose,  2  parts  peptone  (Chapoteaut).  Prepare 
as  for  ordinary  agar,  make  slightly  alkaline  and  to  3  parts  of  the  product  mix  1  part 
of  ox  serum  previously  heated  to  60°  C. 

Broth. — In  early  sub-cultures  either  no  growth  occurs,  or  it  may  be  a  very 
minimal  amount  insufficient  to  render  the  medium  cloudy,  a  few  coherent 
masses  and  a  little  deposit  being  found.  When  acclimatized  the  cultures  may 
become  more  abundant  and  assume  the  same  characteristics  as  in  serum 
broth. 

Serum-broth.  Ascitic-broth. — Coherent  masses  are  first  seen  and  then 
towards  the  third  day  a  greyish,  thin,  delicate  film  appears. 

Rabbit  serum. — A  slight  cloudiness  with  formation  of  coherent  masses. 

Milk  is  not  coagulated  by  the  growth  of  the  organism. 

Potato. — Very  fine,  greyish  deposit. 


SECTION  III.— BIOLOGICAL  PROPERTIES. 
1.   Biochemical  reactions. 

The  Meningococcus  ferments  glucose  and  maltose,  but  has  no  action  on 
laevulose.  saccharose  and  inulin. 

Media  containing  glucose  and  maltose. — If  to  media  containing  glucose  or 
maltose  litmus  solution  or  neutral  red  be  added  to  serve  as  indicators,  such 
media  can  be  used  for  the  diagnosis  of  the  Meningococcus. 

The  Meningococcus  forms  acid  out  of  glucose  and  maltose  and  turns  the 
litmus  red.  To  tubes  of  lactose  agar  add  one-third  its  volume  of  ascitic  fluid 
(p.  53)  and  about  1  c.c.  of  litmus  solution  and  set  in  the  sloped  position. 

Buchanan  recommends  Loeffler's  serum-broth  containing  1  per  cent,  glucose 
and  0*01  per  cent,  of  a  1  per  cent,  solution  of  neutral  red.  This  medium  at 
first  of  a  yellowish  colour  is  turned  pink  in  about  24  hours  by  the  Meningo- 
coccus. Sloped  tubes  of  lactose-ascitic-agar  containing  a  trace  of  neutral  red 
may  also  be  used. 

The  meningococcus  produces  no  indol. 


648  THE   MENINGOCOCCUS 


2.  Vitality  and  virulence. 

Vitality. — At  room  temperature  cultures  die  in  from  4-6  days  :  in  the  ice 
chest  they  become  sterile  in  36-48  hours.  In  early  sub-cultures  the  Meningo- 
coccus  dies  in  3-6  days,  but  after  being  resown  several  times  the  organism 
may  live  in  the  incubator  for  as  long  as  3  months  (Albrecht  and  Ghon). 

Cultures  are  sterilized  in  5  minutes  at  65°  C.,  in  a  few  minutes  at  80°  (X 
and  instantly  at  100°  C.  :  and  are  also  sterilized  by  desiccation  for  3  hours 
at  20°  C. 

Virulence. — The  virulence  of  the  Meningococcus  is  very  inconstant,  different 
strains  presenting  varying  degrees  of  pathogenicity,  and  it  does  not  appear 
to  be  increased  by  passage  through  animals. 

The  severity  of  the  symptoms  produced  in  the  human  subject  by  a  given 
strain  of  Meningococcus  bears  no  relation  to  the  virulence  of  that  strain  for 
the  lower  animals. 

On  account  of  the  considerable  variation  in  the  virulence  of  different 
strains  of  Meningococci  most  observers  use  strains  from  several  sources  in 
immunizing  animals  for  the  preparation  of  antimeningococcal  serum. 

3.  Toxins. 

Cultures  of  the  Meningococcus  sterilized  by  heat  kill  susceptible  animals 
as  easily  as  living  cultures.  Filtered  cultures  have  no  toxic  properties 
(Jundell,  Albrecht  and  Ghon).  Flexner  has  shown  that  an  emulsion  of 
Meningococci  sterilized  with  toluol  and  filtered  to  remove  the  bacteria  is 
toxic. 

Dopter  from  his  experiments  concludes  that  the  toxin  is  an  endotoxin  and 
that  the  Meningococcus  secretes  no  soluble  toxins.  The  toxin  can  be  extracted 
by  autolysis  by  treating  the  bacteria  with  distilled  water  :  solutions  prepared 
in  this  way  are  often  used  for  immunizing  animals  and  preparing  anti- 
meningococcal serum  (vide  infra). 

4.  Immunization.    Serum  therapy. 

Ruppel  immunizes  rabbits  by  inoculating  them  sub-cutaneously  first  with 
a  non-virulent  strain  then  with  a  strain  known  to  be  of  high  and  constant 
virulence.  The  serum  of  these  animals  has  prophylactic  properties  (0'004  c.c. 
protects  against  100  lethal  doses).  Ruppel's  investigations  led  Flexner,  and 
then  other  bacteriologists,  to  attempt  the  immunization  of  horses  for -the 
purpose  of  preparing  a  therapeutic  serum  :  the  various  methods  which  have 
been  employed  will  be  considered  under  three  heads. 

A.  Flexner  immunizes  horses  by  inoculating  them  sub-cutaneously  first 
with  dead  cultures,  then  with  living  cultures,  an$  finally  with  an  autolytic 
extract  obtained  by  making  an  emulsion   of  virulent  cultures  in  water. 
(Flexner  in  his  earlier  experiments  inoculated  the  horses  intra-venously  but 
this  method  was  abandoned  in  favour  of  sub-cutaneous  inoculation.) 

B.  Kolle,  Wassermann  and  Leber  immunize  three  horses  simultaneously. 

One  is  inoculated  with  cultures  of  a  single  strain  of  the  Meningococcus.  The  first 
inoculation  given  sub-cutaneously  consists  of  one-half  a  24-hour  agar  culture  made 
into  an  emulsion  with  normal  saline  solution  and  heated  to  60°  C.  :  the  second  of 
a  whole  culture  similarly  treated  :  the  third  of  one-half  a  living  non-heated  culture 
and  the  fourth  of  a  whole  non-heated  culture.  Afterwards  the  horse  is  inoculated 
intra-venously  on  successive  occasions  with  one-half,  one,  two,  three,  and  four 
living  cultures. 

A  second  horse  is  treated  in  a  similar  manner  but  with  a  mixture  of  5  or  6  strains 
of  Meningococci  from  different  sources. 


SERUM  THERAPY  649 

The  third  horse  is  inoculated  with  toxins.  Young  agar  cultures  in  Roux  bottles 
are  washed  off  with  sterile  distilled  water,  shaken  for  48  hours,  O5  per  cent,  carbolic 
acid  added  and  the  whole  centrifuged.  The  supernatant  liquid  is  toxic  for  guinea- 
pigs  in  doses  varying  from  0*1—1  c.c.  intra-peritoneally.  The  first  inoculation  for 
the  horse  is  a  quantity  equivalent  to  10  times  the  fatal  dose  for  a  guinea-pig.  The 
horse  is  treated  for  a  long  time  with  sub-cutaneous  inoculations  of  the  toxic  extracts 
and  afterwards  by  intra-venous  inoculation  ;  great  care  has  to  be  exercised  in  inocu- 
lating these  extracts  since  symptoms  of  anaphylaxis  are  not  at  all  uncommon. 

The  serum  of  the  three  horses  is  mixed  in  equal  parts  for  therapeutic 
purposes. 

C.  Dopter  immunizes  horses  solely  by  means  of  living  cultures  which  are 
inoculated  first  sub-cutaneously  and  later  intra-venously. 

Properties  of  anti-meningococcal  serum. — The  serums  of  animals  immunized 
by  any  of  the  above  methods  exhibit  agglutinating,  sensitizing,  opsonizing 
and  precipitating  properties.  They  possess  undoubted  therapeutic  properties 
in  meningococcal  cerebro-spinal  meningitis  but  in  no  other  forms  of  meningitis. 

Therapeutics. — In  meningococcal  meningitis  the  serum  should  be  inoculated 
into  the  sub-arachnoidal  space.  Lumbar  puncture  is  first  performed  and  a 
quantity  of  fluid  let  out  equal  to  or  rather  greater  than  the  volume  of  serum 
to  be  inoculated  :  then,  without  withdrawing  the  needle  a  fairly  large  dose 
of  serum  is  inoculated  slowly  (20-40  c.c.  for  an  adult,  15-20  c.c.  for  a  young 
child).  The  inoculation  must  be  repeated  twice,  three  times  and  even  four 
times  at  intervals  of  a  few  days,  indications  for  the  further  use  of  the  serum 
being  shown  by  the  clinical  symptoms  and  more  especially  by  the  appearance 
of  the  cerebro-spinal  fluid.  When  the  latter  becomes  clear  and  limpid,  and 
the  Meningococci  have  disappeared,  and  the  pus  cells  are  replaced  by  normal 
poly-morpho-nuclear  leucocytes  or  lymphocytes,  no  further  administration 
of  serum  is  needed. 

5.  Agglutination. 

The  serum  of  persons  suffering  from  meningococcal  meningitis  generally 
agglutinates  the  Meningococcus  in  a  dilution  of  1  in  100  (Albrecht  and  Ghon : 
Bettencourt  and  Franga).  The  agglutination  would  appear  to  be  better 
marked  with  the  strain  isolated  from  the  patient  than  with  strains  recovered 
from  other  sources  (1  in  50).  Antimeningococcal  serum  also  agglutinates  the 
Meningococcus  in  dilutions  of  1  in  200,  1  in  500  and  even  1  in  1000. 

Note. — The  examination  of  the  agglutination  reaction  is  open  to  two  sources 
of  error. 

I.  The  Meningococcus  is  not  always  agglutinated  by  specific  serums.  And 
Kutscher  observed  that  some  strains  never  agglutinate  at  a  temperature  of  37  °C., 
but  agglutinate  very  well  when  left  at  55°  C.  for  24  hours  :  it  is  advisable  therefore 
when  an  organism  has  been  isolated  having  the  morphological  appearances  of  the 
Meningococcus  but  which  does  not  agglutinate  with  a  specific  serum  at  37°  C.  to  test 
the  reaction  at  55°  C. 

H.  Some  closely  related  organisms  are  agglutinated  by  an  antimeningococcal 
serum.  The  Gonococcus  is  an  interesting  case  in  point.  An  antimeningococcal 
serum  will  agglutinate  both  the  Meningococcus  and — though  more  feebly — the 
Gonococcus,  and  conversely  an  antigonococcal  serum  will  agglutinate  the  Meningo- 
coccus as  well  as  the  Gonococcus.  Dopter  and  Koch  have  however  shown  that  if 
an  emulsion  of  the  Meningococcus  be  added  to  an  antimeningococcal  serum  it  will 
absorb  the  whole  of  the  agglutinin  while  absorption  with  the  Gonococcus  will  not 
remove  the  agglutinin  for  the  Meningococcus  :  if  on  the  other  hand  an  emulsion 
of  the  Gonococcus  be  added  to  an  antimeningococcal  serum,  and  after  the  organism 
is  agglutinated  the  mixture  be  centrifuged,  the  clear  supernatant  fluid  will  agglutinate 
the  Meningococcus  but  will  have  no  such  action  upon  the  Gonococcus.  The  anti- 
meningococcal serum  therefore  contains  specific  agglutinins  for  the  Meningococcus 
and  group  agglutinins  which  act  alike  on  the  Meningococcus  and  on  the  Gonococcus. 

This  experiment  which  demonstrates  differences  of  a  specific  nature  between  the 


650  THE  MENINGOCOCCUS 

Gonococcus  and  Meningococcus  is  further  confirmed  by  the  fact  that  antigono- 
coccal  serum  contains  no  immune  body  (sensibilisatrice)  for  the  Meningococcus  and 
conversely  (Vannod). 

The  agglutination  reaction. — For  the  diagnosis  of  the  Meningococcus  the 
agglutination  reaction  should  be  carried  out  as  follows  :— 

1.  Take  five  narrow  sterile  test-tubes. 

2.  To  the  first  add  1  c.c.  of  a  1  in  100  dilution  of  unheated  antimeningococcal 
serum.     To  the  second  1  c.c.  of  a  1  in  200  dilution  of  the  same  serum.     To 
the  third  1  c.c.  of  a  1  in  100  dilution  of  normal  horse  serum.     To  the  fourth 
1  c.c.  of  a  1  in  200  dilution  of  normal  horse  serum.     To  the  fifth  1  c.c.  of 
normal  saline  solution. 

3.  To  each  tube  add  one  loopful  of  a  young  agar  culture  of  the  orgasnism 
under  investigation. 

4.  Incubate  the  tubes  at  37°  C.  for  24  hours.     (If  no  agglutination  has 
taken  place  repeat  the  experiment  and  incubate  at  55°  C.) 

5.  If  the  organism  be  a  Meningococcus,  on  taking  the  tubes  out  of  the 
incubator  there  will  be  very  distinct  agglutination  in  tubes  Nos.  1  and  2  while 
the  emulsion  in  the  other  three  tubes  will  be  cloudy. 

SECTION  IV.— THE  ISOLATION  AND  IDENTIFICATION  OF  THE 
MENINGOCOCCUS. 

1.  The  diagnosis  of  meningococcal  meningitis. — The  diagnosis  of  meningo- 
coccal  meningitis  should  be  based  upon  the  following  characteristics  of  the 
organism  isolated. 

1.  Microscopical  appearance  :    "  coffee  bean  "  diplococci  situated,  in  the 
case  of  meningeal  exudates,  within  the  leucocytes. 

2.  Staining  reactions  :    gram-negative. 

3.  Cultural  characteristics  :  capacity  to  grow  on  ordinary  agar  :  fermenta- 
tion of  glucose  and  maltose,  no  action  on  Isevulose  and  saccharose. 

4.  Agglutination  by  a  specific  serum. 

2.  The  isolation  of  the  organism. — The  cerebro-spinal  fluid  and  also  the 
exudate  on  the  naso-pharynx  should  be  examined. 

A.  Cerebro-spinal  fluid. — To  establish  a  diagnosis  of  meningococcal 
meningitis  it  is  essential  to  examine  the  fluid  obtained  by  lumbar  puncture. 

In  cases  of  this  disease  the  cerebro-spinal  fluid  is  generally  purulent  or  cloudy : 
but  this  indication,  though  present  according  to  Netter  in  96  per  cent,  of  cases,  is 
not  pathognomonic.  In  the  early  stages  of  the  infection  (during  the  first  24  hours) 
the  cerebro-spinal  fluid  is  clear  and  it  may  again  become  clear  later,  towards  the  end 
of  the  second  week. 

Centrifuge  the  cerebro-spinal  fluid  in  a  sterile  tube  and  then  proceed  as 
follows  : 

1.  Examine  a  portion  of  the  deposit  microscopically.     Stain  a  film  with  a 
simple  stain  and  another  by  Gram's  method. 

2.  Sow  the  remainder  of  the  deposit  on  blood-agar  or  ascitic-agar  and 
incubate  for  24  hours  at  37°  C.     Examine  the  colonies  and  sow  some  on 
glucose  and  on  maltose  tinted  with  litmus  or  neutral  red  (vide  ante]  and  use 
others  for  the  agglutination  reaction. 

Vincent  and  Bellot's  reaction.— Centrifuge  the  cerebro-spinal  fluid  immedi- 
ately after  collection.  Drop  50-100  drops  of  the  clsar  supernatant  fluid  into 
each  of  two  sterile  tubes :  to  one  tube  add  1  or  2  drops  of  antimeningococcal 
serum  and  incubate  for  6-12  hours  at  37°  C.  or  55°  C.  If  the  case  be  one 
of  meningococcal  meningitis  the  tube  to  which  the  antimeningococcal  serum 
has  been  added  is  slightly  cloudy  while  the  control  tube  remains  clear. 


MICROCOCCUS  CATARRHALIS  651 

The  reaction  sometimes  fails  ;  and  occasionally  both  tubes  are  cloudy 
when  it  is  of  course  valueless.  But  in  general  it  is  a  useful  test  especially 
when  meningococci  are  not  found  in  the  cerebro-spinal  fluid. 

Note. — In  a  certain  number  of  cases  (25  per  cent,  according  to  von  Lingels- 
heim)  no  organism  can  be  detected  in  the  cerebro-spinal  fluid  though  they 
can  be  proved  to  be  cases  of  meningococcal  meningitis  by  examining  the 
agglutinating  properties  of  the  blood  and  by  Vincent  and  Bellot's  reaction  : 
and  it  must  be  assumed  either  that  the  organism  was  present  in  the  early 
stages  of  the  disease  and  has  vanished  or  that  it  is  confined  to  the  upper 
part  of  the  cerebro-spinal  axis. 

B.  Naso-pharyngeal  exudates. — The  material  must  be  collected  from  the 
naso-pharynx  from  behind  the  fold  of  the  palate.  A  small  plug  of  sterile 
wool  fixed  on  a  rigid  metal  wire  and  slightly  curved  at  the  end  may  be  used 
to  collect  the  exudate  :  pass  this  wool  plug  as  high  as  possible  behind  the 
palate  and  sow  the  material  without  delay  on  ascitic-agar  plates.  Incubate 
for  24  hours  at  37°  C.  and  carefully  examine  the  colonies  :  any  that  look 
suspicious  must  be  tested  as  described  above.  Confusion  is  most  likely  to 
arise  with  the  Micrococcus  catarrhalis  (vide  infra) ;  this  organism  however 
grows  easily  on  all  the  ordinary  media  and  does  not  ferment  either  glucose 
or  maltose. 

Micrococcus  catarrhalis. 

In  recent  years  German  writers  have  described  the  occurrence  in  some  respiratory 
affections  of  a  gram-negative  organism  morphologically  similar  to  the  Gonococcus 
and  which  they  designate  the  Micrococcus  catarrhalis  (Ghon,  Pfeiffer,  Bezan9on, 
Israel  and  de  Jong). 

This  organism  is  frequently  found  in  man  in  cases  of  bronchitis  and  pneumonia, 
and  in  the  sputum  of  tuberculous  persons  whose  temperature  is  raised.  Some 
writers  have  confused  the  Micrococcus  catarrhalis  with  the  Meningococcus,  /  but  it 
is  easily  differentiated  from  the  latter  by  its  cultural  characteristics. 

The  Micrococcus  catarrhalis  grows  well  on  ordinary  media :  does  not  ferment 
carbohydrates :  and  is  not  agglutinated  by  an  antimeningococcal  serum. 

Experimental  inoculation. — The  Micrococcus  catarrMlis  is  only  slightly  virulent 
for  laboratory  animals.  It  gives  rise  to  small  lesions  of  the  pleura  when  inoculated 
in  very  large  doses  into  the  pleural  cavities  of  guinea-pigs  or  mice :  rabbits  are  not 
susceptible. 

Microscopical  appearance. — In  sputum  the  organism  is  seen  as  irregular  diplococci, 
looking  like  coffee  beans  placed  in  pairs  with  their  flattened  surfaces  adjacent. 
They  may  be  seen  singly  or  in  small  groups,  either  free  or  within  the  leucocytes. 
In  cultures  the  appearance  is  much  the  same.  The  diplococci  are  sometimes  arranged 
in  tetrads  but  more  frequently  in  small  clumps.  Chains  are  never  seen  and  the 
organism  is  not  surrounded  by  a  capsule. 

The  Micrococcus  catarrhalis  is  easily  stained  by  the  basic  aniline  dyes  and  is 
gram-negative. 

Cultural  characteristics. — The  organism  is  aerobic  and  grows  at  20°  C.  but  the 
optimum  temperature  is  37°  C. 

Gelatin. — A  slow  and  scanty  growth  takes  place  at  20°  C.  The  medium  is  not 
liquefied. 

Broth. — In  broth  at  37°  C.  a  slight  cloudiness  and  powdery  deposit  is  formed. 

A  gar. — After  24  hours'  incubation  at  37°  C.  the  growth  consists  of  small,  white, 
irregularly  rounded  colonies.  Later  the  centre  becomes  prominent  and  slightly 
brownish  in  colour  while  the  margins  are  wavy  and  jagged. 

Liquid  rabbit  serum. — The  growth  is  minimal  on  this  medium  and  the  organism 
is  not  capsulated. 

M ilk. — The  medium  is  not  coagulated  as  the  result  of  the  growth  of  the  organism. 

Carbohydrate  media. — No  fermentation. 


PART  III. 
THE  PARASITIC  FUNGI. 


CHAPTER  XLVIII. 
THE   PARASITIC  HYPOMYCETES. 

Section  I. — The  genus  Discomyces,  p.  655. 
/.  Discomyces  bovis,  p.  656. 

The  parasites  of  actinomycosis,  p.  660. 

//.  Discomyces  israeli,  p.  661.  ///.  Discomyces  thibiergi,  p.  661.  IV.  Discomyces 
liquefaciens,  p.  661.  V.  Discomyces  garteni,  p.  661.  VI.  Discomyces  astero'ides, 
p.  662.  VII.  Discomyces  forsteri,  p.  662.  VIII.  Discomyces  rosenbachi,  p.  662. 
IX.  Discomyces  madurce,  p.  662.  X.  Discomyces  freeri,  p.  664.  XI.  Discomyces 
brasiliensis,  p.  665. 

The  parasites  of  mycetoma,  p.  665. 

XII.  Discomyces    minutissimus,    p.     666.     XIII.  Discomyces    farcinicus,    p.     667. 
XIV.  Discomyces  caprce,  p.  668.     XV.  Discomyces  hofmanni,  p.  669.     XVI.  The 
polychrome  discomyces  of  Valle'e,  p.  669. 
Section  II. — The  genus  Malassezia,  p.  669. 

1.  Malassezia  furfur,  p.  669.     2.  Malassezia  tropica,  p.  670.     3.  Malassezia  mac- 
fadyeni,  p.  670.     4.  Malassezia  mansoni,  p.  670. 
Section  III. — The  genus  Trichosporum,  p.  670. 
Section  IV. — The  genus  Coccidioides,  p.  671. 
Section  V. — The  genus  Sporotrichum,  p.  672. 
Section  VI. — The  genus  Oidium,  p.  674. 
Section  VII.— A  fungus  of  unknown  classification :  the  parasite  of  Bursattee,  p.  674.     . 

SECTION  I.— THE   GENUS  DISCOMYCES. 

Parasites  of  the  genus  Discomyces — Streptothrix  (Cohn) — were  formerly 
grouped  with  the  Bacteria,  but  Sauvageau  and  Radais  have  shown  that  they 
really  belong  to  the  family  of  the  Oosporidce  of  the  Hypomycetes.  The  para- 
sitic species  of  the  Discomyces  which  produce  disease  in  man  and  the  lower 
animals'  do  not  however  appear,  as  was  thought,  to  belong  to  the  genus 
Oospora,  and  the  majority  of  observers  now  agree  that  it  is  better  to  accept 
Blanchard's  classification  and  to  group  them  among  the  Oosporidce,  but  with 
the  generic  name  Discomyces  (Rivolta). 

The  Oosporidse  are  fungi  consisting  of  a  branched  septate  mycelium — the 
mycelium  was  for  long  considered  to  be  non-septate  but  Gueguen  has  shown 
that  it  is  in  fact  septate — and  in  which  reproduction  takes  place  by  rows  of 
rounded  conidia. 

It  is  not  uncommon  to  find  parasites  belonging  to  the  Oosporidce  in  the  mouth, 
where  they  may  be  responsible  for  white  patches,  ulcers  and  abscesses  in  the  tonsil 
(Roger,  Bory  and  Sartory).  In  the  sputum  of  persons  suffering  from  various  pul- 
monary diseases  both  Roger  and  Flexner  have  found  similar  parasites. 

The  various  species  of  the  genus  Discomyces  possess  certain  characteristics  in 
common.  They  grow  readily  in  artificial  culture  media,  and  in  liquid  media  give 
rise  to  a  growth  somewhat  resembling  the  leaves  of  the  water  lily ;  the  medium 


656  THE   PARASITIC   HYPOMYCETES 

never  becomes  cloudy.     On  gelatin  they  form  small,  spherical,  star-like  colonies  ; 

their  growth  on  potato  consists  of  hard,  dry,  scaly  masses.  The  appearance  of  a 
culture  however  and  also  the  colour  vary  to  some  extent  with 
the  different  species  and  with  age.  In  old  cultures  on  solid 
media  numerous  aerial  hyphse  bearing  chains  of  conidia  project 
from  the  surface :  during  germination  the  envelope  of  the 
spore  bursts,  and  from  it  there  originates  a  young  filament 
which  in  turn  ramifies.  Reproduction  may  also  take  place 
by  transverse  division  of  the  filaments.  So  that  according 
to  the  age  of  the  culture,  microscopical  examination  may 
show  branched  forms,  streptococcal-like  chains,  or  small 
filaments  closely  resembling  the  avian  tubercle  bacillus.  In 
view  of  this  apparent  pleomorphism  it  is  not  surprising  that 
observers  were  for  a  long  time  led  astray  in  their  investigations 

Minto  the  nature  and  identity  of  these  parasites. 
Organisms  of  the  genus  Discomyces  stain  in  a  manner  similar 
to  that  of  the  tubercle  bacillus. 

I.    DISCOMYCES  BOVIS.     (Hartz  ;  R.  Blanchard.) 

Syn.  Actinomyces  bovis  Bellinger  and  Hartz. — Oospora 
bovis  Sauvageau  and  Radais. — Nocardia  actinomyces 
Trevisan. — [Streptothrix  actinomyces  Rossi  Doria.] 

That  cattle  were  subject  to  a  peculiar  disease  charac- 
FIG.  306.— Fructifica-terized  by  the  formation  of  large,  hard,  sarcomatous 
tionof  an  Oospora.  (After  masses  in  the  tongue  and  iaw  bones  which  had  a  tendency 

SaDouraud.)  -111  i  i  i.inr  i  j_-     * 

to  break  down  and  become  purulent  had  for  a  long  time 
been  recognized,  but  the  cause  of  this  disease  was  unknown  until  Bollinger  and 
Hartz  showed  that  it  was  due  to  a  specific  parasite,  to  which  they  gave  the 
name  Actinomyces  bovis.  Shortly  afterwards  Israel  and  Wolff  found  the  same 
organism  in  the  pus  of  an  empyema  in  the  human  subject.  Since  then  a 
very  considerable  number  of  cases  of  Actinomycosis  in  man  have  been  recorded, 
and  the  disease  can  no  longer  be  regarded  as  a  pathological  curiosity. 

Infection  with  the  parasite  in  man,  as  in  cattle,  may  take  the  form  of  a  swelling 
of  the  jaw  bone,  but  lesions  localized  in  other  tissues  of  the  body  and  even  generalized 
infections  are  frequently  seen,  and  may  so  closely  resemble  tuberculosis  as  to  be 
confounded  with  that  disease.  Infection  of  the  lung  (broncho-pneumonia,  pleurisy) 
and  of  the  peritoneum  is  not  uncommon.  It  is  a  difficult  matter  to  diagnose  the 
disease  on  clinical  evidence  alone  so  that  a  microscopical  examination  of  the  pus, 
sputum,  etc.  is  of  great  assistance. 

Actinomycosis  occurs  also  in  animals  other  than  cattle  ;  it  has  been  found  in  pigs, 
deer,  sheep,  horses,  elephants,  and  other  animals. 

Outside  the  body  Discomyces  bovis  lives  as  a  saprophyte  on  cereals,  and  infection 
of  cattle  is  thus  easily  accounted  for.  Man  may  become  infected  by  handling  corn 
or  by  inhaling  dust  during  threshing  operations.  Delearde  produced  an  osteo- 
sarcoma  in  the  maxilla  of  a  sheep  by  inoculating  the  bone  beneath  the  periosteum 
with  a  grain  of  barley  infected  with  the  parasite. 

1.  Experimental  inoculation. 

The  earlier  experiments  made  with  cultures  grown  aerobically  failed  to 
infect  the  inoculated  animals  (Bostrom).  Israel  and  Wolff,  however,  were 
able  to  infect  rabbits  by  inoculating  them  with  cultures  grown  anaerobically 
[vide  D.  Israeli).  Mertens  inoculated  a  rabbit  in  the  anterior  chamber  of 
the  eye  and  produced  nodules  containing  clubbed  forms  of  the  parasite. 

Inoculations  with  pus  from  foci  of  actinomycosis  in  man  have  often  given 
positive  results  in  guinea-pigs,  rabbits  and  cows  (Israel  and  others).  Rabbits 
die  several  months  after  being  inoculated  sub-cutaneously  or  intra-peritone- 


PARASITES   OF  ACTINOMYCOSIS  657 

ally  ;   in  the  latter  case  masses  of  growth  due  to  the  parasite  are  found  post 
mortem  in  the  peritoneum,  omentum  and  mesentery. 

2.  Morphology.    Detection  of  the  parasite. 

A.  Microscopical  appearance. 

1.  In  the  tissues. — -In  cases  of  actinomycosis  the  pus,  sputum,  or  other  in- 
fected tissues  contain  sulphur  coloured  or  occasionally  whitish  opaque  grains, 
varying  in  size  from  a  lycopodium  spore  to  a  millet  seed.  It  is  these  grains 
which  must  be  searched  for  and  examined  in  a  suspected  case  of  actinomycosis, 
and  it  is  very  important  that  the  pus  or  other  material  should  be  quite  fresh, 
as  the  parasite  rapidly  degenerates.  If  the  grains  be  scarce  and  very  small  it 
is  best  to  spread  a  little  of  the  pus  in  a  thin  layer  on  a  slide,  in  this  way  the 
grains  can  easily  be  seen  and  collected  for  further  investigation. 

If  one  of  the  grains  be  crushed  between  a  slide  and  cover-glass  in  a  drop  of 
glycerin  the  parasite  can  be  readily  and  easily  recognized  (fig.  307).  When 


FIG.  307. — An  actinomycosis  grain.     Unstained  FIG.  308. — Section  through   an   actino- 

preparation  obtained  by  crushing  between  two          mycotic  tubercle.     Gram's  method.     (Oc. 
slides.  II,  obj.  8,  Heich.) 

crushed  in  this  way  the  grain  will  be  seen  to  consist  of  small  mulberry-like 
bodies  composed  of  a  central  mass  of  filaments  closely  fitted  together  with 
numerous  diverging  rays  (a/cn's  =a  ray)  in  many  cases  club-shaped  at  the 
free  end  (yellow  colonies,  clubs,  cross-forms)  (vide  infra). 

The  central  part  consists  of  a  tangled  mass  of  filaments  which  appear  to  be 
branched,  mixed  with  small  swollen  corpuscles  :  here  and  there  a  few  filaments 
will  be  seen  to  project  from  the  centre  and  end  externally  by  the  side  of  the 
clubs :  the  filaments  measure  on  an  average  10-12/x  long,  the  clubs  20-30^ 
long  by  8-10/x  broad.  The  clubs  are  merely  degenerated  forms  of  the  parasite, 
the  result  of  the  cellular  reaction  of  the  tissues  ;  the  wall  of  the  filament 
enlarges  at  the  end,  thus  giving  origin  to  a  swelling  or  club,  in  the  centre 
of  which  the  original  filament  can  be  recognized.  Clubs  are  at  first  oval  in 
shape,  but  later  degenerate  and  assume  an  irregular  outline  and  frequently 
undergo  calcareous  degeneration.  Clubs  are  not  present  in  earJy  lesions, 
and  only  occur  when  the  tissues  of  the  host  are  markedly  resistant ;  often 
indeed  when  the  lesions  caused  by  the  parasite  are  healing  the  filaments 
disappear,  and  only  clubs  are  found. 

In  the  tissues  there  is  an  accumulation  around  the  parasite  of  epithelioid 
cells  with  large  oval  nuclei.  These  cells  are  arranged  in  a  circle  and  may  fuse 
to  form  a  giant  cell  containing  in  addition  to  several  nuclei  the  parasite  itself. 

Staining  reactions. — The  filaments  of  Discomyces  bovis  stain  with  the  basic 
aniline  dyes  as  well  as  by  Gram's  and  Ziehl's  methods,  but  they  are  not  as 

2T 


658  THE   PARASITIC  HYPOMYCETES 

resistant  to  the  decolourizing  action  of  acid  as  the  tubercle  bacillus.  The 
clubs  stain  with  picrocarmine,  safranin  or  eosin.  A  fresh  specimen  stained 
with  picrocarmine  shows  the  clubs  detached  and  stained  yellow  on  a  pink 
background  of  cells. 

Colonies  crushed  between  slides  may  be  stained,  after  drying  and  fixing  in 
alcohol-ether,  by  Gram's  method  and  counter-stained  with  eosin.  The 
filaments  will  be  stained  violet  and  the  clubs  yellow  or  pink  (fig.  308).  Sec- 
tions may  be  stained  in  the  same  manner.  Weigert's  method  may  also  be 
used. 

Equally  pretty  preparations  may  be  obtained  by  staining  sections  for 
30-50  minutes  in  carbol-fuchsin,  decolourizing  rapidly  in  1  per  cent,  sulphuric 
acid,  washing  in  alcohol,  then  in  water,  and  counterstaining  with  an  aqueous 
solution  of  methylene  blue. 

IK-  -'--Vtfr 

•*>\ 


'*?>     S  ~    %*&%#*;'••'  ••&* 

"  f  &  3     £4  *5.#   *JP^  f*St.    ?  *  JW 

^e?&«K&V&* 

;&£*3*if 


FIG.  309. — Discomyces  bovis.     Section  through  a  bovine  lesion.     Carbol-fuchsin 
and  methylene  blue.     (Oc.  2,  obj.  T\th,  Zeiss.) 

Morel  and  Dulaus  recommend  the  following  technique  for  sections :  Stain  for  a 
few  minutes  in  Delafield's  haematoxylin  (acetic  acid  is  added  until  the  solution  has 
a  red  tint).  Wash  in  water.  Treat  for  3  minutes  in  the  following  solution : — 

Victoria  blue,       -  1  gram. 

Alcohol,      -  10  c.c. 

Water,        -  90     „ 

Wash  again.     Treat  with  Gram's  solution  for  a  few  moments.     Wash  in  alcohol. 
Stain  for  a  few  minutes  in  : — 

Rosalin-violet,     -  1  gram. 

Alcohol,  -  10  c.c. 

Water,        -  90     „ 

Wash  in  water  again.  Pass  rapidly  through  absolute  alcohol.  Decolourize 
very  rapidly  in  a  mixture  of  equal  parts  of  essence  of  cinnamon  and  absolute  alcohol. 
When  the  sections  have  acquired  a  red  colour  wash  in  alcohol,  clear  in  xylol  and 
mount  in  balsam.  The  nuclei  of  the  cells  are  violet,  the  mycelium  of  the  parasite 
blue,  the  clubs  bright  red. 

For  staining  pus  when  no  yellow  grains  can  be  found  with  the  naked  eye,  Lemiere 
and  Becue  recommend  the  following  method :  1.  Spread  a  little  of  the  pus  on  a 
slide,  dry  and  wash  in  ether.  2.  Treat  for  a  few  minutes  with  a  30  per  cent,  solu- 
tion of  soda.  3.  Stain  for  a  quarter  of  an  hour  in  a  5  per  cent,  aqueous  solution  of 
eosin.  4.  Wash  in  a  saturated  aqueous  solution  of  sodium  acetate  and  examine 
the  preparation  in  this  solution.  The  centres  of  the  colonies  are  red,  the  clubs 
pale  yellowish-pink. 


PARASITES   OF  ACTINOMYCOSIS  659 

2.  In  cultures. — The  appearance  of  the  parasite  in  cultures  is  quite  different 
from  its  appearance  in  the  tissues.  Branched  filaments,  coccal  forms  and 
short  rods  are  found,  but  no  clubs  are  seen.  In  young  cultures  the  mycelium 


FIG.  310. — Discomyces  bovis.     Section  through  an  human  lesion.    Gram's 
stain  and  eosin.    (Oc.  2,  obj.  J*th,  Zeiss.) 

consists  of  delicate,  branched,  non-septate  filaments  which  are  sometimes 
very  long.  In  older  cultures  these  filaments  divide  into  short  stout  rods 
(bacillary  and  coccal  forms) ;  the  aerial  hyphse  are  thick  and  carry  chains  of 
conidia.  In  very  old  cultures,  swollen  and  irregular-shaped  involution  forms 
are  found. 

B.  Cultural  characteristics. 

Conditions  of  growth. — Discomyces  bcvis  grows  equally  well  under  aerobic 
or  anaerobic  conditions.  Growth  commences  at  20°  C.  is  most  luxuriant 
at  37°  C.,  less  abundant  at  40°  C.,  but  still  continues  up  to  about  50°  C. 
Most  of  the  ordinary  media  are  suitable  for  the  cultivation  of  the  parasite, 
the  best  growths  however  are  obtained  on  serum  and  on  media  containing 
glycerin. 

Some  difficulty  is  often  experienced  in  isolating  the  parasite  from  pus 
because  in  most  cases  it  is  associated  with  the  ordinary  organisms  of  suppura- 
tion, and  these  grow  over  the  culture  medium  before  the  discomyces  has  had 
time  to  start.  There  are  several  methods  of  isolation,  the  most  satisfactory 
being  that  described  by  Bostrom. 

Spread  the  pus  containing  the  yellow  grains  on  plates  of  gelatin  and  incubate 
for  a  couple  of  days.  On  then  examining  the  plates  most  of  the  grains  will  be  sur- 
rounded by  colonies  of  contaminating  organisms  but  a  few  will  be  seen  here  and 
there  which  are  more  or  less  discrete  and  isolated.  Pick  these  off  with  a  stout 
platinum  wire  and  transfer  them  to  tubes  of  coagulated  serum  and  incubate  the 
cultures  at  37°  C.  After  5-6  days  the  colonies  of  discomyces  begin  to  grow.  If 
this  method  be  adopted  it  is  advisable  to  sow  a  good  number  of  tubes  of  serum 
because  many  of  them  will  ultimately  prove  to  be  contaminated. 

Culture  media.  Glycerin-broth. — When  sown  and  incubated  at  37°  C. 
white,  granular,  hemispherical  colonies  appear  in  about  5  or  6  days  and  may 
grow  as  large  as  a  pea.  The  colonies  fall  to  the  bottom  of  the  tube  leaving 
the  medium  quite  clear. 

Coagulated  serum. — After  being  incubated  for  about  5  days,  small  whitish 
or  yellowish  colonies  appear,  dry,  firm,  and  often  confluent. 


660  THE   PARASITIC   HYPOMYCETES 

Glycerin-agar. — Growth  appears  after  2  days  and  takes  the  form  of  small, 
yellowish-white,  dry,  wrinkled  colonies,  firmly  adherent  to  the  medium.  The 
colonies  soon  become  confluent  and  form  a  broad,  yellowish, 
wrinkled  band,  covered  with  rough  projections. 

Gelatin. — Discomyces  bovis  grows  feebly  on  gelatin. 
Liquefaction  takes  place  slowly  and  to  a  very  slight  extent. 
On  plates,  after  incubating  for  about  6  days,  small,  greyish, 
punctiform  colonies  appear  with  yellow  centres  and  irregular 
outline. 

Potato. — After  about  a  week  or  rather  less,  small  colourless 
colonies  appear  ;  a  few  of  these  soon  become  greyish  and 
prominent  then  the  growth  thickens  and  forms  a  yellowish, 
wrinkled,  mammillated  layer  sometimes  edged  with  black. 
The  potato  turns  brown  in  the  neighbourhood  of  the 
growth. 

Milk.— Milk  is  not  coagulated. 

Cultures  on  seeds. — On  fresh  grains  of  corn  or  dry  seeds 
softened  in  water  Discomyces  bovis  grows  as  a  yellowish, 
powdery  layer  and  penetrates  into  the  interior  of  the  seed. 

3.  Biological  properties. 

Vitality. — Discomyces  bovis  is  fairly  resistant  to  heat  arid 
other  destructive  agents.     Cultures  are  killed  by  exposure 
to   a   temperature   of  70°-75°  C.   for   10  minutes   (Wolff), 
^n.       Cultures  on  dried-up  agar  or  gelatin  live  for  more  than  a 

± IG.    oil. — DlSCO-  _^  \     _°  V          ,.         .  ,  /..         -i 

myces  bovis.  Culture  year.  Poncet  found  the  parasite  alive  in  culture  alter  being 
weekg)!ycerin  agar  (1  neglected  for  four  years.  Ordinary  antiseptics  appear  to 
have  little  effect  on  the  organism :  but  on  the  other  hand, 
1  drop  of  a  1  per  cent,  solution  of  methylene  blue  added  to  10  c.c.  of  a  broth 
culture  sterilizes  the  latter. 

Virulence. — The  virulence  of  Discomyces  bovis  is  attenuated  by  passage 
through  the  tissues  of  man  and  some  of  the  lower  animals.  Its  vegetative 
and  pathogenic  properties  can  be  restored  by  growing  it  on  vegetable  tissues 
(Liebmann).  A  plant  which  grew  from  a  seed  inoculated  with  Discomyces 
bovis,  was  found  to  be  infected,  the  parasite  taking  the  form  of  very  short 
filaments,  and  these  were  pathogenic  on  inoculation  into  animals  (Liebmann). 

Toxins. — Filtered  glycerin-broth  cultures  of  Discomyces  bovis  contain  a 
toxin  (Streptothricine,  Delearde)  which,  like  tuberculin,  has  very  little  effect 
on  healthy  animals,  but  in  infected  animals  gives  rise  to  a  febrile  reaction. 

Association  with  other  micro-organisms. — In  the  tissues,  Discomyces  bovis 
is  frequently  found  in  association  with  other  organisms,  most  frequently  with 
the  more  common  organisms  of  suppuration,  and  occasionally  in  pulmonary 
lesions  with  the  tubercle  bacillus.  Sputum  in  which  the  Discomyces  has  been 
found  should  always  be  examined  for  the  tubercle  bacillus. 

The  parasites  of  actinomycosis. 

"^Recent  investigations  would  tend  to  show  that  the  clinical  condition  of  actino- 
mycosis is  not  a  specific  disease  but  may  be  due  either  to  Discomyces  bovis  or  to  one 
of  several  other  parasites  which  though  differing  from  are  all  more  or  less  related  to 
it.  Some  cases  of  actinomycosis,  for  instance,  have  been  shown  to  be  due  to  Dis- 
comyces asteroides  (vide  infra]  which  has  been  found  by  several  observers  in  human 
lesions.  [Discomyces  liquefaciens  and  Discomyces  garteni  (vide  infra)  have  also  been 
isolated  from  cases  of  the  disease.  ] 

Ferre  and  Fajuel,  Scheele  and  Petruschky,  have  described  cases  of  actinomycosis 


PARASITES   OF  ACTINOMYCOSIS  661 

caused  by  a  Discomyces  which  gave  white  cultures  and  did  not  liquefy  gelatin  (Disco- 
myces  alba).  In  a  case  recorded  by  Sabrazes  and  Riviere  the  parasite  was  yellow 
on  cultivation  and  did  not  liquefy  gelatin  (Discomyces  flava).  [D.  alba  and  D.  flava 
are  probably  the  same  species  as  D.  asterotdes.] 

Levy  isolated  an  orange-coloured  Discomyces  from  a  case  of  human  actinomycosis. 
The  parasite  liquefied  gelatin  but  did  not  appear  to  be  pathogenic "  to  the  lower 
animals.  This  is  possibly  the  polychrome  Discomyces  of  Vallee  (vide  infra). 

Lignieres  and  Spitz  have  described  an  actinomycotic  condition  (actino-bacillosis) 
due  to  a  Discomyces  which  is  [said  to  be]  readily  distinguishable  from  the  classical 
Discomyces  bovis  ;  it  grows  in  artificial  culture  as  a  short  bacillus  and  only  rarely 
exhibits  a  filamentous  appearance  (on  potato).  This  parasite,  Discomyces  spitzi, 
is  pathogenic  for  cattle,  sheep,  rabbits  and  guinea-pigs.  According  to  J.  H.  Wright, 
however,  it  is  identical  with  Discomyces  bovis.  [Brumpt  describes  the  organism  as 
a  bacillus  (Actinobacillus  lignieresi  Brumpt).  "  The  actinobacillus  of  Lignieres  and 
the  tubercle  bacillus  (Schlerothrix  kochi  of  Metchnikoff)  are  aberrant  micro-organisms 
which  many  observers  consider  as  fungi  related  to  the  Discomyces  "  (Brumpt).] 


II.   DISCOMYCES  ISRAELI. 

LrSyn. — Streptothrix  israeli  Kruse. — Streptothrix  spitzi  Lignieres. — 
Discomyces  bovis  Brumpt. 

[Discomyces  israeli  closely  resembles  in  its  appearance  in  tissues  and  cultures 
Discomyces  bovis,  and  by  some  observers  the  two  parasites  are  regarded  as  the 
same  species.  The  main  difference  between  them  would  seem  to  be  that 
cultures  of  Discomyces  boris  are  not  inoculable  into  animals  while  Discomyces 
israeli  readily  gives  rise  to  typical  lesions  of  actinomycosis  on  inoculation 
into  animals.  Discomyces  israeli  moreover  grows  better  under  anaerobic 
conditions  than  when  cultivated  in  presence  of  a  free  supply  of  air. 

[Discomyces  israeli  has  been  found  in  cattle  in  the  Argentine  by  Lignieres  and 
Spitz,  in  both  man  and  cattle  by  J.  H.  Wright  in  the  United  States,  and  in  France 
it  would  appear  that  Discomyces  israeli  is  a  more  common  cause  of  actinomycosis  in 
man  than  Discomyces  bovis.] 


III.  DISCOMYCES  THIBIERGI  (Ravaut  and  Pinoy). 

[This  parasite  was  found  by  Ravaut  and  Pinoy  in  a  patient  suffering  from 
disseminated  nodules  beneath  the  skin  and  in  the  muscles. 

[In  the  pus  the  parasite  sometimes  had  the  form  of  isolated  bacilli,  at  other  times 
it  formed  very  small  white  grains  quite  different  from  the  yellow  grains  seen  with 
other  parasites  of  actinomycosis.  In  the  tissues  it  forms  clubs. 

[Discomyces  thibiergi  grows  easily  both  under  aerobic  and  under  anaerobic  con- 
ditions. It  appears  to  be  devoid  of  pathogenic  properties  in  the  rabbit,  guinea-pig, 
white  rat  and  monkey  (Macacus).] 


IV.   DISCOMYCES  LIQUEFACIENS. 

[Discomyces  liquefaciens  was  isolated  from  a  clinical  case  of  actinomycosis.  It 
does  not  form  clubs  in  the  tissues,  is  an  obligatory  aerobe,  grows  well  on  various 
media  and  is  not  pathogenic  to  laboratory  animals  (Brumpt).] 


V.   DISCOMYCES   GARTENI. 

[Discomyces  garteni,  isolated  from  a  clinical  case  of  actinomycosis,  is  an  aerobic 
organism,  and  is  pathogenic  to  certain  animals.     It  forms  no  clubs  (Brumpt).] 


662  THE   PARASITIC   HYPOMYCETES 

VI.   DISCOMYCES   ASTEROIDES   (Eppinger). 

Syn.— Oospora  aster  aides  (Sauvageau  and  Radais),  Nocardia  aster  Mes 
(Trevisan),  Streptothrix  aster  Mes  (Gedoelst). 

This  organism  was  originally  found  by  Eppinger  in  pure  culture  in  the  pus 
of  a  cerebral  abscess  in  a  person  who  had  died  of  cerebro-spinal  meningitis. 
The  same  observer  has  since  found  the  parasite  several  times  in  conditions 
clinically  resembling  tuberculosis.  Almquist  also  discovered  what  was 
apparently  the  same  parasite  in  pus  from  a  case  of  meningitis  :  Schabad, 
and  MacCallum,  have  published  similar  observations. 

Experimental  inoculation. — Discomyces  asteroldes  is  pathogenic  for  rabbits 
and  guinea-pigs.  After  inoculation  the  animals  die  of  a  pseudo-tuberculous 
condition  and  the  parasite  is  found  in  large  numbers  in  the  tubercles. 

Microscopical  appearance. — Discomyces  asterdldes  occurs  in  pus  as  branched 
filaments  about  2/x  broad.  It  forms  no  clubs.  In  cultures  rounded  or  coccal 
forms  (spores)  are  found  as  well  as  small  bacillary  filaments  and  branched 
forms.  In  old  cultures  the  mycelial  protoplasm  is  not  homogeneous  but 
shows  vacuoles,  separated  by  cubical  and  rounded  granulations.  In 
young  cultures  the  filaments  are  frequently  star-shaped — hence  the  name 
asteroiides. 

Discomyces  astero'ides  stains  with  basic  dyes  and  retains  the  violet  in  Gram's 
method  ;  it  stains  with  carbol-fuchsin  but  is  more  readily  decolourized  than 
the  tubercle  bacillus. 

Cultures.— Agar  containing  2  per  cent,  glucose  is  the  most  useful  medium 
on  which  to  grow  the  parasite.  The  colonies  are  warty  in  appearance,  white 
at  first  but  eventually  becoming  red-ochre  in  colour,  while  the  surface  becomes 
wrinkled  and  folded.  The  growth  on  gelatin  is  poor  :  the  medium  is  not 
liquefied. 

VII.   DISCOMYCES   FORSTERI   (Gedoelst). 

Syn. — Oospora  forsteri  (Sauvageau  and  Radais) ,  Nocardia  forsteri 

(Trevisan),  Streptothrix  forsteri  (Cohn). 

Discomyces  forsteri  is  found  in  the  lachrymal  ducts,  where  it  forms  small 
whitish  masses  consisting  of  fine,  slightly  branched  filaments,  more  or  less 
twisted  and  mixed  with  coccal  forms.  Attempts  to  grow  the  organism  have 
failed. 

VIII.   DISCOMYCES   ROSENBACHI   (Kruse). 

Syn. — Streptothrix  rosenbachi. 

This  fungus  was  isolated  by  Rosenbach  from  a  sort  of  sub-acute  erysipelas 
of  the  finger,  which  he  described  as  erysipeloid.  Rosenbach  infected  himself 
with  the  parasite  and  reproduced  the  symptoms  of  the  condition. 

Discomyces  rosenbachi  occurs  as  very  fine  twisted  and  slightly  branched 
filaments  ;  it  grows  readily  on  gelatin  at  20°  C.,  forming  small  grey-brown 
colonies  consisting  of  bundles  of  filaments  arranged  around  a  more  dense 
centre. 

IX.   DISCOMYCES   MADURA. 

8301. — Oospora  madurw. — Nocardia  madurce. — Streptothrix  madurce  Vincent. 

If  one  of  the  small  nodes  characteristic  of  the  disease  known  as  Mycetoma 
or  "  Madura  foot "  be  incised  and  squeezed  a  sanious  pus  exudes  which  con- 
tains small,  yellowish-white  grains,  resembling  the  grains  of  actinomycosis. 


PARASITES   OF  MYCETOMA  663 

In  size  the  grains  vary  from  a  millet  seed  to  a  pin's  head  and  consist  of 
innumerable,  closely  interwoven  hyphse. 

1.  Microscopical  appearance. — Under  the  microscope  the  grains  will  be 
found  to  be  composed  of  very  slender  closely  interwoven  filaments  straight 
or  wavy  and  measuring  l/u-l'5/x  across.     In  the  thinner  parts  of  the  film  the 
filaments  appear  to  be  branched  and  at  the 

periphery  of  the  tangled  mycelial  masses  there 
is  a  tendency  to  a  radiating  arrangement. 
Small  irregular  swellings  measuring  about  2/x 
are  often  seen  at  the  extremity  or  in  the  length 
of  the  filaments,  but  clubs  are  never  seen.  All 
these  details  can  be  made  out  by  staining  with 
methylene  blue  or  dilute  carbol-fuchsin  and 
examining  under  a  magnification  of  400  or  500 
diameters. 

In  cultures  the  same  arrangement  is  seen  but 
the  filaments  are  more  slender  and  their  breadth 
does  not  exceed  1/x.  In  cultures  two  weeks 

old  the  ends  of  the  filaments  are  often  broken 

.  i  ,  .  ,  FIG.  312.— Discomyces  madurce. 

up  into  regular,   ovoid   segments   which  are  (After  Vincent.) 

larger  than  the  filaments  themselves,  and  con- 
stitute the  fertile  hyphae.  The  spores  are  refractile,  oval  in  shape,  and  have 
sharply  defined  outlines.  They  measure  T5/X-2/A  broad  and  are  variously 
arranged  in  pairs,  groups  of  three,  chains  or  in  large  masses.  When  sown  in 
a  new  tube  of  broth  they  elongate  at  one  end,  giving  origin  to  a  short  rod 
with  rounded  ends. 

Staining  reactions. — Discomyces  madurce  stains  readily  with  the  basic 
aniline  dyes  and  retains  the  violet  in  Gram's  method.  Eosin  and  safranin 
stain  the  parasite  feebly,  iodine  colours  it  yellow,  hsematoxylin,  violet.  The 
spores  stain  well  with  the  basic  dyes  and  by  Gram's  method. 

Sections. — Excise  some  small  pieces  of  skin  containing  either  the  young, 
hard,  painful  nodules  or  nodules  which  are  softening.  Examination  of  the 
material  is  rendered  somewhat  difficult  on  account  of  the  ease  with  which  the 
grains  drop  out  of  the  tissues.  Vincent  recommends  the  following  technique  : 
Harden  the  pieces  of  skin  successively  in  60  per  cent.,  80  per  cent.,  90  per  cent, 
and  absolute  alcohol.  Embed  in  paraffin.  Fix  the  sections  on  slides  and 
stain  with  Orth's  alcohol  carmine  and  Gram's  stain. 

Under  the  microscope  the  whole  of  the  diseased  area  is  seen  to  form  a  large 
tubercle  in  the  centre  of  which  is  a  mycelial  mass  having  the  characteristics 
described  above. 

2.  Cultural  characteristics. — Discomyces  madurce  grows  at  all  temperatures 
between  20°  and  40°  C.  but  best  at  37°  C.     It  is  a  strict  aerobe.     Growth  is 
always  very  small  in  amount  on  ordinary  media  :   vegetable  infusions  are  the 
most  useful  for  growing  cultures  of  the  organism. 

To  isolate  the  parasite  in  pure  culture  sterilize  the  surface  of  the  skin, 
incise  one  of  the  nodes  with  a  sterile  bistoury,  introduce  a  fine  pipette  through 
the  incision,  and  aspirate  the  contents  of  the  tumour.  Sow  the  material  on 
one  or  other  of  the  following  media. 

Culture  media.  Vegetable  infusions. — An  infusion  (15  grams  to  the  litre) 
of  straw  or  hay  (the  aromatic  plants  must  be  removed)  forms  an  excellent 
medium,  as  does  an  infusion  of  potato  (20  grams  to  the  litre).  Cultures  are 
best  sown  in  Erlenmeyer  flasks,  on  account  of  the  free  supply  of  air. 

After  incubating  for  4  days  at  37°  C.  small  greyish  flocculi  appear,  some  of 


664  THE   PARASITIC  HYPOMYCETES 

which  adhere  to  the  sides  of  the  flask  while  others  fall  to  the  bottom.  After 
3  weeks'  growth  these  flakes  have  attained  the  size  of  a  green  pea  :  some  turn 
brown  in  the  centre  ;  others,  on  the  sides  of  the  flask  or  on  the  surface  of  the 
medium,  become  pink  or  red  in  colour  after  a  month  or  two.  The  liquid  never 
becomes  cloudy,  but  the  surface  is  often  covered  with  a  white  efflorescence 
formed  by  the  spores. 

Meat  broth. — The  growth  on  this  medium  is  very  scanty.  After  incubating 
for  a  fortnight,  small,  rounded,  greyish  granules  are  formed,  the  liquid 
remaining  clear.  After  sub-culturing  several  times  in  broth  the  growth 
becomes  more  abundant. 

Gelatin. — In  ordinary  gelatin  a  very  scanty  white  growth  forms  along  the 
line  of  the  stab  and  on  the  surface  of  the  medium.  Growth  is  more  abundant 
in  the  following  medium  : 

Potato,  or  hay,  infusion  (vide  ante),         -  -         100  c.c. 

Gelatin,       -          -  9  grams. 

Glycerin,     -  4       ,, 

Glucose,      ...  4       ,, 

Neutralize.     Sterilize. 

Discomyces  madurce  does  not  liquefy  gelatin. 

Glucose-glycerin-agar. — Ordinary  agar  is  not  at  all  a  suitable  medium  for 
the  cultivation  of  the  parasite,  but  on  glucose-glycerin-agar  it  grows  freely. 
The  culture  on  the  latter  medium  consists  of  circular,  smooth,  raised  colonies 
yellowish-white  at  first,  becoming  pink  and  even  bright  red  later,  though  the 
colour  eventually  disappears.  When  the  colonies  do  not  coalesce  they 
become  very  large  and  umbilicated,  the  central  depression  being  white  while 
the  raised  margins  are  reddish. 

Potato. — After  incubating  for  5  days  at  37°  C.  small  whitish  projections 
are  seen,  and  these  later  assume  a  mulberry-like  appearance.  Around  the 
growth  the  potato  is  depressed,  but  it  does  not  change  colour.  When  incu- 
bated for  a  month  the  colonies  are  pale  pink  in  colour  and  in  places  the  colour 
deepens  and  ultimately  becomes  bright  red,  orange  or  deep  red.  The  more 
acid  the  potato  the  more  intense  the  colour.  Some  of  the  colonies  appear 
powdered  with  a  fine  whitish  dust  which  consists  of  spores.  Some  potatoes 
are  unsuitable  for  the  growth  of  the  organism. 

Milk. — Growth  takes  places  without  coagulation  of  the  medium. 

Serum.     Egg. — No  growth  takes  place  on  these  media. 

3.  Biological  properties. — All  attempts  to  inoculate  animals  have  failed 
(Vincent  and  Nocard). 

Discomyces  madurce  is  very  resistant  to  drying  ;  cultures  dried  on  sterile 
blotting  paper  for  9  months  have  subsequently  given  a  growth  when  sown 
on  culture  media.  A  culture  on  potato  21  months  old  was  still  alive.  Non- 
spore-bearing  cultures  are  killed  in  from  3-5  minutes  at  a  temperature  of 
60°  C.  Spores  resist  a  temperature  of  75°  C.  for  5  minutes  but  are  killed  in 
3  minutes  at  a  temperature  of  85°  C. 

Associated  micro-organisms. — In  suppurating  nodules  opening  externally 
Vincent  found  besides  the  Discomyces,  Staphylococcus  aureus  and  S.  albus. 


X.   DISCOMYCES  FREERI. 

Syn. — Streptothrix  freeri. 

[This  parasite  was  found  by  Musgrave  and  Clegg  in  a  mycetoma  of  the 
foot  in  a  native  woman  in  Manilla. 

[It  grows  freely  on  artificial  media  but  only  under  aerobic  conditions. 


PARASITES   OF  MYCETOMA  665 

[By  inoculating  pus  from  the  lesion  and  pure  cultures  of  the  organism 
Musgrave  and  Clegg  reproduced  a  typical  mycetoma  in  the  feet  of  three 
monkeys  (Macacus  philippinensis).  Intra-peritoneal  inoculation  proved 
fatal  to  monkeys,  dogs  and  guinea-pigs.  Sub-cutaneous  inoculation  was 
not  followed  by  generalization  of  the  disease.] 


XI.  DISCOMYCES  BRASILIENSIS. 

[Discomyces  brasiliensis  was  isolated  by  Lindenberg  from  a  case  of  myce- 
toma. It  is  strictly  aerobic  ;  it  grows  poorly  at  37°  C.  but  at  ordinary  room 
temperature  grows  well  on  all  ordinary  media.  The  organism  does  not 
appear  to  be  pathogenic  to  rabbits,  guinea-pigs  and  pigeons.] 

The  parasites  of  Mycetoma. 

Syn.— Madura  foot. — [Actinomycosis. — Pseudo-actinomycosis.] 

The  endemic  disease  of  warm  climates  for  a  long  time  inappropriately 
described  as  Madura  foot,  and  now  at  Laveran's  suggestion  known  as  Mycetoma, 
comprises  several  clinical  varieties  :  the  white  or  ochroid,  the  black  or  melanoid 
and  the  red.  As  the  result  of  the  investigations  of  Vincent,  mycetoma  was 
considered  to  be  due  solely  to  an  infection  with  a  single  species  of  Discomyces 
(D.  madurce  Vincent)  but  more  recent  researches  have  shown  that  though 
infection  with  D.  madurce  may  be  the  commonest  cause  of  mycetoma,  many 
cases  are  caused  by  other  and  very  different  species  of  fungi. 

The  white  variety  is  due  to  the  following  parasites  stated  in  the  order  of 
the  frequency  with  which  they  occur,  viz. : — Discomyces  madurce,  [Indiella 
somaliensis,  which  is  perhaps  even  more  common  in  India  than  D.  madurce 
(Manson)],  Sterygmatocystis  nidulans,  Indiella  mansoni,  Indiella  reynieri,  and 
occasionally  Discomyces  bovis  (actinomycotic  mycetoma),  [Discomyces  freeri 
and  Discomyces  brasiliensis]. 

The  melanoid  variety  is  caused  by  two  parasites,  Aspergillus  bouffardi  and 
Madurella  mycetomi,  which  though  provisionally  regarded  as  distinct  are 
probably  identical  species. 

In  a  case  of  mycetoma  of  the  red  variety,  Laveran  and  Pelletieri  found 
zoogloea  masses  of  pink  micrococci  (M.  pelletieri). 

Aspergillary  mycetomata.  White  varieties. — Nicolle  has  recorded  a  case 
of  mycetoma  in  Tunis  due  to  Sterygmatocystis  (Aspergillus)  nidulans  (p.  699). 
The  grains,  yellowish-white  in  colour,  some  as  large  as  a  pea,  were  more  or 
less  spherical  and  had  a  smooth  surface.  Microscopically  they  consisted  of 
large  septate  mycelial  filaments. 

[Indiella  somaliensis l  is  described  by  Brumpt  as  being  the  infecting  agent 
in  two  cases  of  white  mycetoma  observed  by  Bouffard  in  Somaliland.  This 
form  of  the  disease  appears  to  be  very  wide-spread  in  India.] 

Indiella  mansoni1  described  by  Brumpt  in  a  case  of  mycetoma  in  India 
and  Indiella  reynieri  found  by  Reynier  in  a  case  in  Paris  appear  to  be  closely 
related  to  Sterygmatocystis  nidulans. 

Melanoid  variety. — Cases  of  melanoid  mycetoma  characterized  by  the 
presence  of  lesions  with  small,  brittle,  irregular-shaped  black  grains  have 
been  reported  from  Africa  (French  Soudan  arid  Senegal),  Italy  [and  India], 
and  were  due  to  a  species  of  Aspergillus,1  Madurella  mycetomi  (Laveran). 

[*•  The  provisional  genera  Indiella  and  Madurella  are  classified  by  Brumpt  with  the 
Hypomycetes.  These  parasites  have  certain  affinities  with  the  genera  Aspergillus  and 
Sterygmatocystis  (Brumpt).] 


666 


THE   PARASITIC   HYPOMYCETES 


By  some  observers  classi- 
fied with  Ascomycetes 
by  others  with  Hypo- 
mycetes, 


probably 
identical 


The  parasite — Aspergillus  bouffardi — found  by  Bouffard  in  cases  of  mycetoma 
in  Djibouti  and  India  is  apparently  identical  with  Madurella  mycetomi.  These 
parasites  have  not  yet  been  grown  in  artificial  culture. 

[Classification  of  the  parasites  of  mycetoma.] 

[1.  Species  of  Ascomycetes,     -  Sterygmatocystis  nidulans 

Nicolle's  white  mycetoma. 

(Aspergillus  bouffardi 
I      Bouffard's  black  mycetoma. 
1  Madurella  mycetomi 
\     Classical  black  mycetoma. 
Indiella  mansoni 

Brumpt's  white  mycetoma. 
Indiella  reynieri 

Reynier's  white  mycetoma. 
Indiella  somaliensis 

Bouffard's  white  mycetoma. 

(Discomyces  madurce 

Vincent's  white  mycetoma. 
Discomyces  bovis 

Actinomycotic  mycetoma. 
[2.  Species  of  Hypomycetes,  -  -      -j  Discomyces  freeri 

Musgrave  and  Clegg's  white 
mycetoma. 

Discomyces  brasiliensis 
Lindenberg's  white  mycetoma. 

[3.  Micrococcus  pelletieri,       -  The  red  mycetoma.] 


XII.  DISCOMYCES  MINUTISSIMUS. 

Syn. — Microsporum  minutissimum. 

The  parasite  of  erythrasma  was  described  by  Burchardt  under  the  name 
Microsporum  minutissimum.  This  parasite  should  be  placed  among  the 
Oosporidse  as  a  species  of  the  genus  Discomyces. 

Detection. — The  same  methods  are  available  for  the  detection  of  D.  minutis- 
simus  as  will  be  described  for  M alas sezia  furfur.  Sabouraud  recommends  the 
following  technique.  Treat  the  scales  with  ether,  then  with  glacial  acetic 
acid,  wash  in  absolute  alcohol,  stain  with  Unna's  blue,  carbol-thionin  or 
Gram's  stain,  pass  through  alcohol  and  xylol  and  mount  in  balsam. 

Microscopical  appearance. — The  parasite,  which  is  present  in  considerable 
numbers  in  the  corneal  layer  of  the  epidermis,  consists  of  a  long,  delicate, 
wavy,  tangled  and  branched  mycelium,  divided  into  segments  which  are 
arranged  end  to  end  and  often  separated  from  one  another  in  such  a  way  as 
to  resemble  bacilli :  the  filaments  occasionally  end  in  a  cluster  of  very  small 
rounded  spores. 

Cultures. — According  to  De  Michele,  D.  minutissimus  grows  easily  on 
ordinary  media,  producing  on  gelatin  a  brownish,  and  on  potato  a  wine-red 
layer  of  growth.  Man  can  be  inoculated  with  cultures  if  the  skin  be  first 
scratched  with  a  lancet.  Ducrey  and  Reale  dispute  De  Michele's  conclusions  : 
they  consider  that  the  cultures  used  by  that  observer  were  not  cultures  of  the 
parasite  of  erythrasma  at  all.  In  their  opinion  D.  minutissimus  grows  with 


PARASITE   OF  BOVINE   FARCY  667 

considerable  difficulty  on  ordinary  media  between  25°  and  30°  C.,  and  gives 
a  white  growth  on  agar  and  gelatin,  and  a  reddish-brown  growth  on  potato. 

XIII.  DISCOMYCES  FARCINICUS   (Nocard). 

Syn. — Nocardia  farcinica. — Oospora  farcinica. — [Streptothrix  farcinicus.] 
This  species  of  Discomyces,  described  by  Nocard,  is  not  pathogenic  to  man. 
Bovine  farcy  only  affects  cattle  and  must  be  carefully  distinguished  from  farcy 
due  to  the  glanders  bacillus  which  occurs  in  man  and  the  horse.     It  is  characterized 
by  adenitis  and  superficial  lymphangitis  followed  later  by  lesions  of  the  lungs  and 
viscera.     Discomyces  farcinicus  is  probably  to  be  found  in  stable-Utter  and  soil, 
and  it  appears  likely  that  animals  become  infected  through  some  solution  of  con- 
tinuity of  the  integuments.     In  Guadeloupe  the  disease  is  thought  to  be  transmitted 
by  a  tick  of  the  family  Ixodidae  (Hyalomma  cegyptium). 

1.  Experimental  inoculation. 

The  parasite  of  bovine  farcy  is  inoculable  into  cattle,  sheep  and  guinea-pigs. 
Guinea-pigs  are  the  most  suitable  animals  for  experimental  purposes.  Rab- 
bits, horses  and  dogs  are  immune. 

In  guinea-pigs  sub-cutaneous  inoculation  leads  to  the  formation  of  an 
enormous  abscess  complicated  by  lymphangitis.  The  abscess  ultimately 
discharges  externally  and  the  animal  recovers. 

Intra-peritoneal  inoculation  leads  in  2  or  3  weeks  to  a  condition  resembling 
tuberculous  peritonitis  :  the  omentum  and  the  surfaces  of  the  abdominal 
viscera  are  covered  with  tubercle-like  nodules. 

Intra-venous  inoculation  is  rapidly  fatal  and  produces  a  true  generalized 
miliary  tuberculous-like  condition.  All  the  viscera  are  infiltrated  with 
miliary  granulations. 

2.  Morphology  and  methods  of  detection. 

(a)  Microscopical  appearance. — The  fungus  of  bovine  farcy  occurs  as 
delicate  filaments  twisted  into  clusters  from  the  periphery  of  which  numerous 
prolongations  take  origin,  giving  an  appearance  very  like  that  of  the  seeds 
of  burrs.  The  filaments  are  not  much  branched. 
"  Clubs  "  are  never  seen. 

In  cultures  numerous  very  small  oval  spores  are 
found,  which  do  not  stain  by  ordinary  methods. 

Staining  reactions. — The  organism  stains  with 
the  basic  aniline  dyes  and  is  gram-positive. 

(/?)  Methods  of  detection. — Films  should  be 
prepared  with  the  pus  and,  after  staining  by 
Gram's  method  and  eosin,  examined  for  the 
parasite. 

For  sections,  harden  the  pseudo-tuberculous 
lesions  in  alcohol,  embed  in  paraffin  and  stain 
by  Gram's  method,  using  eosin  or  Orth's  picro- 
carmine  as  a  ground  stain.  The  clusters  of  FIG.  sis.— Discomyces  farcinicus. 
filaments  will  be  found  in  the  centres  of  the  3^  (8l$"$.  9?^*™*'* 
tubercles. 

(7)  Cultural  characteristics. — Discomyces  farcinicus  is  a  strictly  aerobic 
organism  and  grows  on  ordinary  media  when  incubated  at  between  30°  and 
40°  C.  Pure  cultures  can  be  easily  obtained  by  removing  with  a  Pasteur 
pipette  some  of  the  material  from  the  centre  of  an  abscess  which  has  not 
opened  externally. 


668  THE   PARASITIC  HYPOMYCETES 

Culture  media.  Broth. — Whitish  irregular  flakes  appear,  some  of  which 
float  on  the  surface  and  form  a  greyish  dusty  pellicle  while  others  fall  to  the 
bottom.  The  medium  remains  clear. 

Glycerin-broth. — A  similar  but  more  luxuriant  growth. 

Agar.— Small,  rounded,  raised,  opaque,  yellowish-white  colonies  develop, 
which  coalesce  to  form  a  mammillated,  folded,  dull,  dusty-looking  culture. 

Serum. — The  growth  has  the  same  characteristics  as  on  agar  but  is  less 
abundant. 

Potato. — An  abundant  growth  develops  consisting  of  considerably  raised, 
dry,  scaly,  yellowish  plaques  with  sharp  cut  edges. 

Milk.— The  growth  assumes  the  form  of  small  greyish  granules.  The 
medium  is  not  coagulated. 

XIV.  DISCOMYCES   CAPR^l   (Gedoelst). 

Syn. — Streptothrix  caprce  Silberschmidt. 

This  parasite  was  found  in  the  lung  of  a  goat  affected  with  pseudo- 
tuberculosis. 

1.  Experimental  inoculation. 

Cultures  of  Discomyces  caprce  are  virulent  for  rabbits  and  guinea-pigs  : 
white  mice  are  susceptible  but  to  a  lesser  degree. 

Sub-cutaneous  inoculation  into  rabbits  and  guinea-pigs  leads  to  the  forma- 
tion of  an  abscess  ;  inoculated  intra-venously  the  parasite  produces  tubercles 
in  the  internal  organs.  "  On  histological  examination  these  tubercles  show 
a  structure  similar  to  that  of  true  tubercles  due  to  the  tubercle  bacillus. 
Giant  cells  are  found  in  the  lungs.  The  tubercles  rapidly  caseate  "  (Silber- 
schmidt). 

2.  Morphology. 

(a)  Microscopical  appearance. — The  mycelium  consists  of  very  fine,  more 
or  less  branched  filaments  of  varying  length.  In  the  tissues  the  longer  forms 
predominate  :  in  agar  cultures,  on  the  other  hand,  and  in  colonies  growing 
on  the  surface  of  broth,  short  rod-shaped  forms  are  the  most  noticeable 
feature. 

Discomyces  caprce  is  non-motile  :  in  cultures  it  forms  elongated  spores 
which  are  readily  decolourized  and  are  only  slightly  resistant  to  heat. 

Staining  reactions. — Discomyces  caprce  stains  with  the  basic  dyes  con- 
taining a  mordant :  it  is  gram-positive.  In  the  tissues  the  organism  is  more 
difficult  to  stain  than  in  cultures  :  use  Gram's  stain  with  eosin  as  a  counter- 
stain. 

(/?)  Cultural  characteristics. — Discomyces  caprce  is  almost  strictly  aerobic. 
It  grows  at  ordinary  temperatures  (but  best  at  33°-37°  C.)  on  the  ordinary 
culture  media,  but  particularly  well  on  2  per  cent,  glucose  broth  and  on 
potato. 

Culture  media.  Gelatin. — Gelatin  is  not  liquefied.  In  plate  culture,  the 
colonies  develop  slowly  and  resemble  small  colonies  of  moulds.  In  stab 
culture,  discrete  flocculent  colonies  develop  in  the  depth  of  the  medium, 
while  on  the  surface  the  growth  takes  the  form  of  a  dry  brownish  layer. 

Glycerin-agar. — A  dry  brownish  layer  appears,  subsequently  sprinkled 
with  white.  The  culture  grows  into  the  agar  in  the  form  of  fine  radiating 
prolongations.  Occasionally  the  colonies  have  a  crater-like  depression  in  the 
centre. 

Broth. — Sugar  broth  is  better  than  ordinary  broth.  Growth  is  visible  after 
about  48  hours  in  the  warm  incubator  (37°  C.)  ;  the  medium  is  clear  ;  small 


PARASITE   OF  PITYRIASIS  VERSICOLOR  669 

colonies  like  thin  concave  discs,  dry  and  whitish,  have  grown  on  the  surface. 
Later  they  become  confluent,  cover  the  whole  of  the  surface  of  the  liquid  and 
climb  up  the  sides  of  the  vessel.  Some  of  the  colonies  fall  to  the  bottom  of 
the  tube  and  form  a  rather  scanty  deposit. 

Potato. — On  potato,  a  thin  whitish  growth  appears  and  later  becomes 
prominent,  brownishrpink  in  colour  and  sprinkled  with  white. 

Milk. — The  surface  of  the  medium  is  covered  with  a  pinkish- white  growth. 
The  milk  is  not  coagulated. 


XV.  DISCOMYCES  HOFMANNI. 

Syn. — Oospora  hofmanni. — Nocardia  hofmanni  Trevisan. — Micromyces 

hofmanni  Max  Griiber. 

Max  Griiber  isolated  from  air  a  streptothrix,  Discomyces  hofmanni,  which 
is  very  like  Discomyces  bovis,  but  when  inoculated  into  rabbits  produces  a 
local  abscess  which  resolves  spontaneously.  Growth  in  artificial  culture 
media  begins  at  22°  C.  :  glucose-agar  is  the  best  medium  :  no  growth  takes 
place  on  potato  and  ordinary  gelatin. 


XVI.    THE  POLYCHROME  DISCOMYCES  OF  VALL^E. 

This  streptothrix,  found  by  Vallee  in  the  blood  of  an  horse  which  had  died 
of  an  acute  pasteurellosis,  does  not  infect  either  laboratory  animals  or  the 
larger  animals,  but  in  broth  cultures  forms  a  toxin  which  is  fatal  to  rabbits 
and  guinea-pigs. 

It  is  a  strict  aerobe  and  grows  on  all  the  ordinary  culture  media.  Cultures 
on  peptone  media  are  salmon  red  :  on  glycerin  media,  yellow  :  on  potato 
the  organism  forms  a  pellicle  which  is  at  first  pinkish-grey,  and,  as  it  becomes 
older,  red. 

SECTION  n.— THE   GENUS   MALASSEZIA. 
1.  Malassezia  furfur. 

Syn. — Microsporum  furfur. 

Pityriasis  versicolor  (Tinea  versicolor)  is  due  to  a  fungus,  Malassezia  furfur, 
discovered  by  Eichstedt. 

Methods  of  examination. — Detach  a  few  of  the  epithelial  scales  from  a 
patch  of  pityriasis  by  lightly  scraping  the  latter  with  the  edge  of  a  slide, 
soak  them  in  a  few  drops  of  a  warm  40  per  cent,  solution  of  potash  on  a  slide 
and  examine  them  in  the  solution  (p.  690).  The  scales  may  also  be  treated 
with  acetic  acid  and  mounted  in  glycerin  tinted  with  eosin. 

Masses  of  the  fungus  will  be  seen  lying  in  the  interstices  between  the 
epithelial  cells.  The  parasite  consists  of  mycelial  filaments  and  rounded 
corpuscles  :  the  corpuscles  are  spherical,  measuring  3-5/x  in  diameter,  and  are 
enclosed  in  a  cuticle  of  cellulose  arranged  spirally  :  the  mycelial  filaments 
are  short,  measuring  3-4/u  in  diameter,  septate,  somewhat  wavy,  slightly 
branched,  sometimes  placed  end  to  end  and  often  bent  on  themselves  in  the 
form  of  a  V. 

Very  little  is  known  of  the  development  of  Malassezia  furfur. 

Cultures. — Malassezia  furfur  is  a  difficult  organism  to  grow  though  cultures 
have  been  obtained  by  Spielschka  and  by  Matzenauer.  Media  containing 
glycerin  are  the  best  for  the  purpose  (Kotliar).  On  glycerin-agar  at  37°  C. 
small  wrinkled  pale  yellow  colonies  are  formed,  and  these  may  attain  the 


670 


THE   PARASITIC   HYPOMYCETES 


size  of  a  pin's  head.     On  gelatin,  growth  is  very  slow, 
tufted  almost  translucent  flocculi  appear. 


In  broth,  small  white 


FIG.  314. — M alassezia  furfur. 

Experimental  inoculation. — Positive  results  have  been  obtained  by  inocu- 
lating cultures  on  the  human  arm  (Spielschka,  Matzenauer). 

In  rabbits,  by  rubbing  a  culture  of  the  fungus  into  a  shaved  area  of  the 
skin  and  protecting  the  inoculated  part  with  a  dressing,  characteristic  patches 
are  formed  in  about  a  week. 

Species  of  malassezia  found  in  the  tropics. 

[2.  Malassezia  tropica. — M.  tropica  is  the  infecting  agent  in  Pityriasis 
versicolor  flava  (Tinea  rosea)  a  common  disease  in  Ceylon.  The  fungus  has 
a  thick,  irregular,  constricted  mycelium. 

[3.  Malassezia  macfadyeni.— This  fungus  is  the  cause  of  another  mycotic 
pityriasis  in  Ceylon,  Pityriasis  versicolor  alba.  The  fungus  has  a  short, 
slender,  straight  mycelium. 

[4.  Malassezia  mansoni.  (Syn.  :  Mierosporon  mansoni). — This  species  is 
the  cause  of  Pityriasis  versicolor  nigra,  a  variety  described  many  years  ago  by 
Manson  as  occurring  in  South  China.  The  parasite  contains  much  dark 
pigment  in  the  mycelial  tubes,  and  in  culture  in  maltose-agar  produces  black 
hemispherical  colonies.  It  is  very  common  in  Ceylon.] 


SECTION  III.— THE   GENUS  TRICHOSPORUM. 

Parasites  of  the  genus  Trichosporum  are  fungi  which  grow  on  the  hair  of  the 
head,  beard  and  moustache  and  form  nodosities  of  firm  but  variable  consist- 
ence. The  parasite  was  first  found  in  Colombia,  South  America,  on  the  hair 
of  a  woman  affected  with  Piedra}-  Cases  of  Trichosporosis  have  since  been 

[*  Piedro,  a  stone  :  from  the  consistence  of  the  nodosities,  which  though  very  firm 
are  not  so  hard  as  the  name  would  indicate  (Manson).  ] 


PARASITES   OF  TRICHOSPOROSIS  671 

described  in   Europe   in   which    the   hair    of    the    head    and    beard    was 
attacked. 

In  trichosporosis  the  interior  of  the  hair  is  never  affected,  but  on  the 
surface  the  parasite  forms  irregular  nodosities  which  more  or  less  completely 
surround  it. 

These  little  nodosities  consist  of  an  amorphous  substance  containing  short 
or  elongated  cells  which  from  mutual  pressure  are 
polyhedral :  after  dissociation  in  potash  the 
greater  number  of  the  cells  are  seen  to  be  placed 
end  to  end  in  branched  chains.  In  cultures  on  solid 
media  these  cells  elongate  and  form  true  filaments. 

Fungi  of  the  genus  Trichosporum  grow  easily 
on  agar,  gelatin,  broth,  potato,  Raulin's  and 
other  media. 

Several  species  have  been  described  :  Tricho- 
sporum giganteum,  the  cause  of  Piedra  in  Colombia, 
which  forms  very  firm  nodosities  (Behrend)  ; 
Trichosporum  ovo'ides  (Behrend),  Trichosporum 
ovale  (Unna),  Trichosporum  beigeli  (Beigel,  Vuille- 
min)  found  on  the  hair  of  the  beard  and  moustache 
in  Europe.  [Trichosporum  krusi  and  Trichosporum 
foxi  are  species  found  by  Castellani  in  Ceylon.] 

SECTION  IV.— THE  GENUS  COCCIDIOIDES. 

These  parasites  are  still  little  known.  They  were  first  found  in  man  by 
Wernicke  in  Buenos-Ayres  in  a  case  of  dermatitis  (cancerous  dermatitis, 
Guiart),  afterwards  by  Rixford  and  Gilchrist  in  the  United  States,  by  Posadas 
in  Argentina  and  has  also  been  seen  in  San  Francisco. 

The  disease  beginning  in  the  skin  more  or  less  rapidly  infects  the  lymphatics, 
becomes  generalized,  and  after  a  variable  length  of  time  terminates  fatally  ; 
it  can  be  reproduced  in  mammals  and  birds  by  sub-cutaneous  inoculation, 
monkeys  being  very  susceptible  to  infection  (Posadas).  In  the  neighbourhood 
of  the  infected  spots,  the  skin  becomes  covered  with  papules  ;  these  unite  and 
form  plaques  the  centres  of  which  ulcerate  and  discharge  a  purulent  fluid 
containing  numerous  cysts  ;  in  the  glands  and  internal  organs,  the  lesions 
are  similar  to  those  in  miliary  tuberculosis. 

In  the  lesions  of  the  skin  the  parasite  is  not  found  within  the  epithelial 
cells  but  in  tubercles  similar  to  the  tubercles  of  actinomycosis  (giant  cell 
formation  containing  the  Coccidioides  and  surrounded  by  epithelioid  cells). 
The  parasite  is  present  in  large  numbers  in  the  pus. 

Blanchard  considers  that  the  various  parasitic  forms  which  have  been 
described  are  all  identical  (Coccidioides  immitis),  though  other  observers 
regard  them  as  constituting  three  different  species.  Blanchard  originally 
classified  the  parasite  with  the  Coccidia  among  the  Sporozoa.  Since  then, 
however,  Buschke,  Ophiils,  Cohn,  have  shown  that  it  will  grow  on  agar, 
giving  origin  to  mycelial  filaments  and  budding  forms  :  it  therefore  becomes 
necessary  to  transfer  the  parasite  to  the  fungi,  and  it  is  possibly  closely  allied 
to  the  genus  Oidium. 

C.  immitis  consists  of  spherical  corpuscles,  20-80/x  in  diameter,  enclosed 
within  a  thick  cuticle.  It  grows  easily  on  agar  and  the  cultures  on  inoculation 
will  infect  susceptible  animals.  Both  in  the  tissues  and  in  cultures  some  of 
the  spherical  bodies  appear  to  contain  spores  which  are  set  free  by  dehiscence 
of  the  enveloping  membrane. 


672 


THE   PARASITIC   HYPOMYCETES 


SECTION  V.— THE   GENUS   SPOROTRICHUM. 

A  disease,  characterized  chiefly  by  the  presence  of  "  chronic  sub-cutaneous 
abscesses  "  or  "  multiple  disseminated  gummata  "  which  in  the  course  of  5  or 
6  weeks  soften  and  break  down,  was  first  described  as  occurring  in  man  by 
Schenk.  Since  then  cases  have  been  recorded  by  Hektoen  and  Perkins,  de 
Beurmann,  and  by  Ramond  and  Matruchot. 

This  disease  has  been  shown  to  be  due  to  a  fungus  to  which  Smith  gave 
the  name  Sporotrichum.  Two  species  were  originally  described — S.  schenki 
(Hektoen  and  Perkins),  and  S.  beurmanni  (Ramond  and  Matruchot) — but 
they  appear  to  be  identical  and  should  be  regarded  as  one,  viz.  :  S.  schenki. 
Numerous  cases  of  sporotrichosis  have  now  been  studied  by,  among  others, 
Dor  (S.  dori  ?),  de  Beurmann  and  Gougerot,  Nattan-Larrier  and  Loeper,  etc. 
The  parasite  may  infect  the  buccal,  pharyngeal  and  laryngeal  mucous  mem- 
branes, may  produce  "  gummata  "  in  the  muscles  and  in  the  mammary  gland, 
and  also  papular  and  vesicular  dermatitis,  osteitis,  synovitis  and  adenitis. 

1.  Morphology  and  methods  of  detection. 

To  demonstrate  the  parasite  it  is  best  to  collect  some  pus  from  a  non- 
ulcerated  "  gumma  "  with  a  sterile  syringe,  using  a  needle  of  large  calibre 
and  adopting  the  necessary  precautions  to  prevent  contamination.  A  por- 
tion of  the  material  should  be  examined  microscopically  after  staining  with 
Unna's  blue,  and  some  should  be  sown  on  Sabouraud's  glucose-agar  (vide 
infra). 

(a)  Microscopical  appearances. — In  pus,  the  Sporotrichum  has  the  appear- 
ance of  a  yeast  and  consists  of  oval  or  fusiform  bodies  representing  spores  or 
conidia  and  measuring  3-6/x  x  2-4/x. 


FIG.  316. — Culture  of  Sporotriclmm.     (After  Monier-Vinard.) 

To  study  the  morphology  of  the  parasite  the  slide  cultivation  method  of 
de  Beurmann  and  Gougerot  is  the  best.  Pour  a  little  Sabouraud's  agar  (vide 
infra)  into  a  large  tube,  and  stand  two  or  three  pairs  of  slides  separated  by 
little  pieces  of  cork  vertically  in  the  tube  :  only  the  lower  ends  of  the  slides 
should  touch  the  agar.  Sterilize  in  the  autoclave  and  then  by  tilting  the 
tube  run  the  melted  agar  over  the  surfaces  of  the  slides.  Sow  the  thin  film 


THE  PARASITE   OF  SPOROTRICHOSIS  673 

of  agar  deposited  on  the  slides  by  lightly  touching  it  with  a  platinum  needle 
charged  with  pus.  Small  colonies  soon  appear  on  the  slides  if  kept  at  the 
ordinary  temperature  and  they  can  be  examined  directly. 

The  colonies  consist  of  a  mycelium  and  spores. 

The  spreading  mycelium  is  formed  of  long,  delicate,  colourless,  septate 
and  branched  filaments  measuring  about  2/x  across. 

The  spores  or  conidia  are  brown,  oval  or  fusiform  (3-6ft  x  2— 4/x)  and  though 
occasionally  arranged  round  the  mycelial  filaments,  are  more  frequently 
collected  in  clusters  of  3  to  30  on  the  end  of  the  filaments. 

In  some  sugar  media  no  mycelium  is  seen,  and  the  parasite  then  assumes 
the  yeast-like  appearance  which  it  has  in  the  tissues. 

Staining  reactions. — The  Sporotrichum  stains  easily  with  the  aniline  dyes 
and  particularly  well  with  Unna's  blue  :  haematoxylin  is  useful  for  staining 
the  spores.  The  parasite  stains  irregularly  with  Gram's  stain,  portions  only  of 
the  mycelium  retaining  the  violet.  The  spores  are,  to  some  extent,  acid-fast. 

(/3)  Cultures. — The  Sporotrichum  grows  only  in  presence  of  air  :  on  ordinary 
media  the  growth  is  poor  but  on  media  containing  sugar  or  glycerin  it  grows 
luxuriantly.  Sabouraud's  agar  is  the  most  suitable  medium  : 

Water,  -       1000  c.c. 

Peptone,     -  10  grams. 

Crude  glucose,  -  40      „ 

Agar,  18       „ 

The  organism  grows  at  any  temperatures  between  12°-39°  CL  but  best 
between  20°  and  30°  C. 

Sabouraud's  agar. — Sow  1  c.c.  of  pus  on  the  surface  of  the  medium  and 
leave  at  the  temperature  of  the  laboratory.  De  Beurmann  and  Gougerot 
adopt  this  method  of  cultivation  as  a  means  of  diagnosis. 

In  from  4r-10  days  a  characteristic  growth  develops  :  at  first  a  dull  spot, 
about  the  sixth  day  it  becomes  whitish  streaked  with  blue,  dry  and  convex  ; 
later  the  colony  folds  on  itself  like  the  convolutions  of  the  brain  and  is  sur- 
rounded by  a  shiny  areola  ;  after  about  12  days  to  3  weeks  the  colour  changes 
to  brown  and  then  to  brownish-black,  while  the  areola  remains  white  and 
becomes  covered  with  a  white  dust. 

If  at  the  same  time  as  the  surface  of  the  medium  is  sown  the  dry  wall  of 
the  tube  be  also  sown  the  organism  grows  on  the  glass,  and  can  be  examined 
in  that  situation. 

Glucose-broth. — Growth  takes  the  form  of  white  pellicles  which  are  formed 
one  after  another  each  in  turn  sinking  to  the  bottom  ;  occasionally  in  old 
cultures  the  pellicle  is  brown.  The  broth  remains  clear. 

Glycerin-carrot.  Glycerin-beetroot. — White  colonies  appear  in  about  3 
days  ;  these  rapidly  coalesce  to  form  a  layer  which  is  at  first  white  and  later 
brown  or  even  black,  the  growth  at  the  same  time  becoming  folded  and 
looking  as  though  dusted  with  powder. 

2.  Experimental  inoculation. 

Eats  and  white  mice  are  particularly  susceptible.  Sub-cutaneous  inocula- 
tion is  followed  by  the  formation  of  an  abscess  at  the  site  of  inoculation, 
which  subsequently  softens  and  ulcerates  ;  then  a  number  of  sub-cutaneous 
"  gummata  "  appear,  osteo-arthritis  develops  in  several  joints  and  occasionally 
abscesses  form  in  the  lungs,  liver  and  spleen. 

Intra-peritoneal  inoculation  leads  to  an  acute  miliary  pseudo-tuberculosis 
or  to  lesions  of  sub-acute  pseudo-tuberculosis.  In  male  rats  a  double  orchitis 
is  a  common  complication  (de  Beurmann,  Gougerot  and  Vaucher). 

Monkeys  are  also  susceptible.     Rabbits,  newly-born  guinea  pigs,  dogs  and 


674 


THE   PARASITIC   HYPOMYCETES 


paper. 


newly-born  cats  can  also  be  infected  (gummata,  pulmonary  tubercles,  granular 

lesions). 

3.  Serum  diagnosis. 

The  serum  of  persons  suffering  from  sporotrichosis  agglutinates  the  spores 
of  the  parasite  (sporo-agglutination  of  Widal  and  Abrami). 

For  the  purposes  of  the  reaction  take  a  portion  of  an  one  to  three-months' - 
old  culture  on  Sabouraud's  medium,  break  it  up  dry  in  a  mortar,  make  an 
emulsion  with  the  powder  in  a  little  normal  saline  solution  and  filter  through 

Mix  the  filtrate  with  the  serum  to  be  tested. 

tinder  these  conditions  the  serum  from  a  case  of  sporotrichosis  agglutinates 
the  spores  in  50-60  minutes  when  diluted  400  to  500  times.  Normal  serum 
has  no  agglutinating  action.  The  serum  of  persons  suffering  from  actinomy- 
cosis  occasionally  agglutinates  the  spores  of  Sporotrichum  but  only  when 
much  less  highly  diluted — 1-60  at  most  (group-agglutination). 

Complement  fixation.— The  serum  of  persons  suffering  from  sporotrichosis 

contains  specific  immune  bodies  (sensibili- 
satrices)  (Widal  and  Abrami,  Joltrain 
and  Weil,  Brissaud).  This  can  be  shown 
by  the  ordinary  methods  of  complement 
fixation  (p.  233). 

SECTION  VI.— THE  GENUS  OIDIUM. 

Q       The    genus    Oidium    includes    several 
g,  parasitic   species   of  phanerogamic   vege- 
table organisms. 

One  saprophytic  species,  Oidium  lactis, 
is  very  widely  distributed  and  forms  greyish 
mucous  spots :  the  fungus  consists  of 
elongated  cells  placed  end  to  end  :  the 
terminal  cells  of  the  chains  carry  rows  of 
spores  :  numerous  cells  can  be  seen  in  the 
act  of  budding. 

Under  the  name  Oidium  subtile  cutis 
Babes  has  described  a  fungus  which  he  found  on  certain  ulcers  in  a  woman. 
He  was  able  to  reproduce  similar  lesions  in  rabbits  (p.  701,  also  p.  704). 


FIG.  3i7.-Oid       lactis. 


SECTION  VII.—  [OF  UNKNOWN  CLASSIFICATION.] 

The  parasite  of  the  disease  Bursattee,  or  Leeches. 

The  disease  of  horses,  mules  and  cattle  in  the  United  States  and  of  horses 
in  India,  characterized  by  the  formation  of  nodules  and  known  in  the  United 
States  as  Leeches  and  in  India  as  Bursattee,  is  caused  by  a  fungus  discovered 
by  Steel  and  described  by  F.  Smith. 

In  the  nodosites  irregularly  branched  and  occasionally  swollen  filaments 
are  found.  Around  the  periphery  of  the  latter  small  spherical  bodies  (spores  ?) 
are  frequently  seen,  and  in  the  meshes  of  the  mycelial  network  rounded  disc- 
like  bodies  are  found  the  significance  of  which  is  quite  unknown.  The  parasite 
has  never  been  grown  in  artificial  culture.  Animals  cannot  be  infected  by 
inoculation. 

To  prepare  microscopical  preparations,  dissociate  the  nodules  by  soaking 
in  a  10  per  cent,  solution  of  caustic  potash  in  the  cold  for  12-24  hours.  Sec- 
tions should  be  stained  with  methylene  blue  and  eosin  or  with  the  Ehrlich- 
Biondi  mixture. 


CHAPTER  XLIX. 
PARASITES  OF  THE  FAMILY  MUCORACIDJE. 

Introduction. — General  methods  of  examination,  cultivation,  etc. 
Section  I. — The  genus  Mucor,  p.  676. 
Section  II. — The  genus  Lichtheimia,  p.  677. 
Section  III. — The  genus  Rhizomucor,  p.  678. 
Section  IV. — The  genus  Rhizopus,  p.  678. 

THE  Mucoracidse,  or  moulds,1  are  phycomycetous  fungi  of  very  wide 
distribution. 

The  Mucoracidse  are  characterized  by  a  non-septate  mycelium  carrying 
spore-bearing  hyphse.  Under  anaerobic  conditions,  however,  the  mycelium 
breaks  up  into  very  short  septa  resembling  yeasts.  Reproduction  takes 
place  either  sexually  or  asexual  ly  ;  in  the  former  case  by  means  of  zygospores 
and  in  the  latter  by  means  of  sporangia. 

For  a  long  time  the  Mucoracidse  were  regarded  as  purely  saprophytic 
organisms,  but  in  recent  years  it  has  been  recognized  that  they  play  a  part 
in  human  and  comparative  pathology. 

1.  Microscopical  examination  of  infected  tissues. — Prepare  films  with  the 
pus  or  other  material  and  stain  with  methylene  blue,  thionin,  or  gentian- 
violet.     It  is  often  better  to  examine  the  material  fresh. 

For  histological  purposes,  cut  sections  of  the  tissue  and  stain  with  hsema- 
toxylin,  wash,  counterstain  with  eosin,  dehydrate  and  mount  in  balsam  or 
dammar  resin. 

2.  Cultures. — The  Mucoracidse  grow  best  on  an  acid  medium.     They  can 
be  easily  cultivated  on  slices  of  fruit  or  potato  and  on  pieces  of  sterile  bread. 
A  simple  method  is  to  cut  a  slice  of  bread  into  small  pieces  leaving  the  crust 
on  one  side,  and  place  them  in  potato  tubes  with  a  little  water  at  the  bottom, 
then  plug  the  tube  with  wool  and  sterilize  at  115°-120°  C. 

Decoctions  made  of  dried  fruit,  hay,  yeast,  or  beer-wort,  and  Nsegeli's, 
Raulin's  or  Sabouraud's  media  either  as  liquid  or  after  solidification  with 
gelatin,  are  all  useful  for  growing  the  species  of  this  family. 

3.  Isolation  of  the  fungus. — According  to  Gedoelst  the  most  satisfactory  method 
of  getting  a  pure  culture  from  a  single  spore  is  to  dip  a  sporangium  into  a  watch- 
glass  containing  a  little  sterile  water.     The  sporangium  bursts  immediately  and  the 
spores  are  set  free  in  the  water ;   leave  them  in  the  water  to  swell  for  a  few  hours. 
Then  with  a  platinum  loop  take  up  a  drop  of  the  water,  spread  it  on  a  sterile  slide 

1  The  term  "  mould "  as  generally  used  is  applied  somewhat  loosely,  and  includes 
beside  the  Mucoracidse  a  number  of  fungi  belonging  to  the  order  of  the  Ascomycetes. 
These  will  be  considered  later. 


676  THE  PARASITIC  MOULDS 

and  examine  it  to  see  that  it  does  not  contain  more  than  one  spore  ;  if  there  should 
be  more  than  one  blot  up  some  of  the  water  with  sterile  filter  paper  until  the  drop 
which  remains  contains  only  a  single  spore.  Now  place  a  drop  of  nutrient  medium 
on  the  -preparation  and  by  arranging  a  moist  chamber  the  growth  of  the  fungus  can 
be  studied  under  the  microscope. 

4.  Microscopical  examination  of  cultures.— In  making  microscopical  pre- 
parations of  cultures  certain  precautions  must  be  taken.     The  simplest  method 
is  to  cut  off  a  small  piece  of  the  mould  with  a  pair  of  fine  scissors  and  transfer 
it  to  a  drop  of  alcohol  on  a  slide — water  must  not  be  used  because  not  only 
does  it  fail  to  wet  the  fungus  properly  but  it  also  causes  the  sporangium  to 
burst — cover  with  a  cover-glass  and  run  in  a  drop  of  glycerin.     This  is  easily 
done  by  placing  a  drop  of  glycerin  at  one  edge  of  the  cover-glass  and  drawing 
it  through  with  a  fragment  of  blotting  paper  held  at  the  opposite  edge. — 
Another  method  is  to  place  the  mould  in  a  drop  of  0*5  per  cent,  osmic  acid 
on  a  slide,  leave  for  a  few  minutes,  wash  in  alcohol  then  in  distilled  water 
and  finally  mount  in  glycerin.     The  preparation  may,  if  necessary,  be  stained 
in  an  aqueous  solution  of  safranin  after  soaking  in  osmic  acid. 

The  following  is  the  method  recommended  by  Salomonsen  when  it  is  desired 
to  examine  a  whole  colony.  Transfer  a  young  colony  to  a  slide,  cover  gently 
with  a  cover-glass,  allow  it  to  dry  for  a  few  minutes  and  then  place  a  large 
drop  of  osmic  acid  solution  at  one  edge  of  the  cover-glass.  When,  after  a 
few  minutes,  the  osmic  acid  has  thoroughly  penetrated  the  preparation,  blot 
up  the  excess  and  run  first  a  drop  of  water  and  then  a  drop  of  glycerin 
under  the  cover-glass. 

5.  Inoculation  experiments. — Attention  was  first  drawn  to  the  pathogenic 
properties  of  some  of  the  Mucors  (the  white  moulds)  by  Lichtheim  and  by 
Lindt  and  the  observations  of  these  investigators  have  been  confirmed  by 
Lucet  and  Costantin. 

The  severity  of  the  disease  following  inoculation  of  these  fungi  depends 
not  so  much  upon  the  virulence  of  the  parasite  as  upon  the  number  of  indi- 
viduals inoculated  ;  and  herein  they  present  a  great  contrast  to  the  bacteria. 
This  may  be  explained  by  the  fact  that  though  the  spores  germinate  when 
introduced  into  the  tissues,  reproduction  has  never  been  known  to  occur  in 
the  body.  Direct  transmission  of  the  parasite  from  animal  to  animal  has 
never  been  observed  ;  when  infection  occurs,  it  is  the  result  of  the  inocula- 
tion of  spores.  It  is  not  possible  to  produce  an  infection  by  direct  inoculation 
of  portions  of  the  mycelium,  and  before  infection  of  an  healthy  animal  can 
be  effected  spores  must  have  been  formed  outside  the  tissues  of  the  living 
body. 

A  disease  fatal  in  a  few  days  follows  the  inoculation  of  the  spores  of  Lich- 
theimia  corymbifera,  Mucor  pusillus,  etc.  into  the  veins  or  peritoneal  cavities 
of  rabbits  ;  and  when  the  tissues  are  examined  post  mortem  numerous  mycelial 
threads  will  be  found  in  the  kidneys  (which  show  lesions  of  nephritis),  Peyer's 
patches  and  also  in  the  lungs  (Lichtheim,  Barthelat).  The  inoculation  of 
Rhizomucor  parasiticus  into  the  veins  of  rabbits,  guinea-pigs  or  fowls  also 
leads  to  a  fatal  disease  (Lucet  and  Costantin). 


SECTION  I.— THE   GENUS  MUCOR. 
Mucor  mucedo. 

Mucor  mucedo  is  one  of  the  moulds  most  commonly  found  in  food-stuffs 
and  other  organic  matter.  It  grows  luxuriantly  producing  tall,  whitish, 
woolly-looking  tufts.  The  mycelium  is  branched  and  gives  rise  to  tall  spore- 
bearing  hyphae  or  pedicels,  each  of  which  is  swollen  at  its  distal  end  into  a 


MYCOSIS 


677 


columella,  and  around  every  columella  a  large  bristly  sporangium  is  formed 
(fig.  318  S.).  The  sporangium  opens  after  the  manner  of  an  hinged  soap 
box  and  sets  free  the  rounded  spores  contained  within  it. 

Two  cases  of  human  pulmonary  mycosis  have  been  attributed  to  this 
parasite  by  Furbinger  (but  see  L.  corymbifera).  And  according  to  Hess, 
M.  mucedo  is  the  cause  of  a  fatal  disease  in  bees — muscardine.  [It  is  also 
pathogenic  for  fish.  ]  On  rabbits  and  guinea-pigs  the  inoculation  of  M.  mucedo 
has  no  effect  (Barthelat). 


C- 


FIG.  318.— Mucor  mucedo.    P,  pedicel 
C,  columella  :  S,  sporangium. 


FIG.  319. — Lichtheimia  racemosa.  A,  raceme- 
like  sporangia.  (After  Fischer.)  B,  yeast-like 
mycelium. 


[SECTION  II.— THE   GENUS  LICHTHEIMIA.] 
1.  Lichtheimia  racemosa. 

Syn. — Mucor  racemosus. 

This  again  is  a  widely  distributed  species.  The  spore-bearing  hyphse  are 
straight  and  irregularly  branched,  the  branches  being  short,  simple  and 
ending  in  sporangia.  L.  racemosa  is  not  pathogenic  to  guinea-pigs  or  rabbits. 
Several  cases  of  pulmonary  mycosis  observed  in  birds  were,  however,  believed 
by  Bollinger  to  be  due  to  this  organism. 

2.  Lichtheimia  corymbifera. 

Syn. — Mucor  corymbifer. 

The  pathogenic  properties  of  this  fungus  are  better  known  than  those  of 
the  other  moulds.  Morphologically,  it  is  differentiated  from  the  preceding 
species  by  the  fact  that  its  flat  hyphse  are  indistinguishable  by  the  naked 
eye  from  the  thick  white  mycelium.  The  hyphse  carry  several  sporangia 
arranged  in  a  corymb. 

In  the  majority  of  the  experimental  inoculations  with  the  Mucoracidse  this 
has  been  the  species  inoculated.  It  is  pathogenic  for  rabbits  (vide  ante). 
It  has  been  found  in  man  in  the  ear  (auricular  mucor-mycosis)  and  in  the 
pharynx  (naso-pharyngeal  mucor-mycosis)  (Siebenham,  Hiickel  and  others). 
One  case  of  generalized  mucor-mycosis  in  man  in  which  the  symptoms  were 
of  a  typhoid  nature  was  attributed  to  this  parasite  (Paltauf)  ;  and  it  would 
seem  that  the  two  cases  of  human  mycosis  (pulmonary  mucor-mycosis) 
described  by  Furbinger  and  referred  to  above  should  be  attributed  to  this 


678 


THE  PARASITIC  MOULDS 


species  rather  than  to  M.  mucedo.  It  has  been  recorded  also  in  association 
with  a  Tricophyton  parasite  in  the  epidermal  scales  of  the  horse  (Lucet  and 
Costantin). 

SECTION  III.— THE   GENUS  RHIZOMUCOR. 
Rhizomucor  parasiticus. 

This  species  was  found  by  Lucet  and  Costantin  in  the  sputum  of  a  woman 
suffering  from  a  condition  resembling  tuberculosis. 

In  cultures,  it  gives  rise  to  a  mycelium  grey  at  first  and  later  fawn-coloured 
with  erect  aerial  mucor-hyphae  or  stolons.  The  fertile  pedicels  are  branched 
and  form  a  raceme  or,  more  rarely,  a  corymb. 

R.  parasiticus  is  pathogenic  to  man  [pulmonary  rhizomucor-mycosis], 
rabbits,  guinea-pigs  and  fowls. 


FIG.  320. — Rhizomucor  varaidticus. 
(After  Lucet  and  Costantin.) 


FIG.  321. — Rhizopus  nigricans.     P,  pedicel ;  C,  columella 
S,  sporangium. 


SECTION  IV.— THE   GENUS  RHIZOPUS. 
1.  Rhizopus  nigricans. 

Syn. — Ascophora  nigricans. 

R.  nigricans  is,  according  to  Megnin,  a  dangerous  fungus,  being  responsible 
for  most  of  the  cases  of  illness  following  the  consumption  of  mouldy  foods. 
It  does  not  seem,  however,  to  be  pathogenic. 

Morphology. — R.  nigricans  forms  blackish  spots  which  consist  of  a  very 
freely-growing,  blackish-brown,  highly-branched  mycelium  with  internodes 
carrying  spore-bearing  hyphae  terminating  in  globular  sporangia.  When 
the  sporangia  (fig.  321  S.)  burst,  the  envelope  becomes  inverted  and  the  ovoid 
or  irregularly  rounded  spores  contained  within  it  are  set  free. 

2.  Rhizopus  niger  seems  to  be  merely  a  variety  of  R.  nigricans.     It  has 
been  recorded  by  Ciaglinski  and  Hewelke  in  certain  cases  of  black  tongue 
(p.  706). 

3.  Rhizopus  cohni  was  found  in  the  rabbit  by  Lichtheim.     Its  characteristic 
feature  is  the  colour  of  the  mycelium,  which  is  at  first  white  but  later  assumes 
a  mouse-grey  colour.     It  is  pathogenic  to  rabbits. 

4.  Rhizopus  equini  was  found  in  the  horse  by  Lucet  and  Costantin.     It  is 
pathogenic  to  rabbits. 


CHAPTER  L. 
PARASITES  OF  THE  FAMILY  GYMNOASCID^. 

Section  I. — The  genus  Tricophyton. 

General  methods  of  examination,  cultivation,  etc. 

A.  Endothrix  species,  p.  682. 

1.  T.  tonsurans,  p.  682.     2.  T.  sabouraudi,  p.  684.     Other  species,  p.  684. 

B.  Ecto-endothrix  species,  p.  685. 

1.  T.  mentagrophytes,  p.  685.     Other  species,  p.  687. 
Section  II. — The  genus  Epidermophyton,  p.  688. 
Section  III. — The  genus  Microsporum,  p.  688. 

1.  M.  audouini. 
Section  IV. — The  genus  Achorion,  p.  690. 

A.  The  human  parasite, — A.  schosnleini,  p.  690. 

B.  The  parasites  of  favus  in  the  lower  animals,  p.  692. 
Section  V. — The  genus  Lophophyton,  p.  692. 

Section  VI. — Micro-organisms  in  Alopecia  areata,  p.  692. 
Section  VII. — The  bacillus  of  Seborrhcea  oleosa,  p.  692. 

[THE  family  of  the  Gymnoascidse  comprises  many  parasitic  fungi.  They  are 
characterized  by  their  conidial  apparatus  and  by  the  fact  that  the  asci  are 
surrounded  by  a  loosely  felted  perithecium.] 

SECTION  I.— THE   GENUS  TRICOPHYTON. 

Griiby  of  Paris  in  1842  was  the  first  to  demonstrate  the  presence  of  fungi 
in  different  forms  of  ringworm.  Malmsten  shortly  afterwards  independently 
described  a  parasite  he  had  found  in  ringworm,  [and  the  name  Tricophyton 
is  of  his  introduction]. 

Formerly,  parasites  of  the  genus  Tricophyton  were  classified  with  the  Bothrytis. 
They  are  now  grouped  with  the  genus  Achorion  in  the  family  Gymnoascidse  (order 
Ascomycetes).  The  Tricophyta  are  closely  related  to  Microsporum  audouini  (Tri- 
cophyton microsporum,  Sabouraud)  another  species  found  in  ringworm  by  Griiby. 
In  cultures  the  Tricophyta  produce  spore- bearing  hyphse  arranged  in  a  raceme 
(conidial  forms). 

The  investigations  of  Sabouraud  have  shown  that  a  number  of  species  of  the 
genus  Tricophyton  is  responsible  for  the  ringworms  of  man  and  the  lower  animals. 

In  man,  the  Tricophyta  infect  the  scalp  (Tinea  tonsurans},  the  beard  (Tinea 
sycosis  vel  barbce),  the  glabrous  skin  (Tinea  circinata),  the  nails  (ony- 
chomycosis),  [and  certain  of  the  mucous  membranes  (mouth  and  vulva)]. 

Methods  applicable  to  the  Tricophyton  parasites  generally. 

1.  Microscopical  examination. — The  infected  hairs  should  be  examined 
after  treating  them  with  a  40  per  cent,  solution  of  potash  in  the  warm  (p.  690). 


680 


THE  PARASITES   OF  RINGWORM 


One  preparation  should  be  gently  heated  to  show  the  situation  of  the  parasite 
in  relation  to  the  hair.  Another  should  be  heated  until  the  liquid  just  begins 
to  boil  in  order  to  dissociate  the  hair  and  show  the  structure  of  the  parasite. 


FIG.  322. — Culture  of  the  white  tricophyton  from  an  horse. 
(After  Bodin.) 


Conidia. 


Epithelial  scales  should  be  first  teased  with  needles  and  then  treated  as 
above.  For  preparing  stained  preparations,  Sabouraud  selects  thin  scales, 
which  should  be  washed  with  chloroform  to  remove  the  fat,  then  boiled  for 
2  or  3  minutes  in  formic  acid,  washed  in  distilled  water  and  stained  in  Sahli's 
borax  blue  for  1  minute. 


*r  ?IQV323/T~A  ringworm-infected  hair  after  treatment  with  caustic  potash. 
JNpte  the  fringe  at  the  junction  of  the  shaft  with  the  soft  bulb.  (From  a 
photograph  kindly  lent  by  Dr.  H.  G.  Adamson  ) 


CULTURE  MEDIA  681 

Sahli's  borax  blue. 

Distilled  water,    -  40  parts. 

Saturated  aqueous  solution  of  methylene  blue,          -  24       „ 

5  per  cent,  solution  of  sodium  borate,     -  15       „ 

After  staining,  the  preparation  is  differentiated  in  absolute  alcohol,  washed 
in  xylol  and  mounted  in  balsam. 

An  hanging-drop  preparation  is  the  best  method  of  studying  the  cultural 
characteristics  of  the  parasites. 

Place  a  drop  of  culture  fluid  on  a  slide,  sow  it  with  the  parasite  and  then  invert 
the  slide  on  a  Bcettcher  cell  in  such  a  way  that  the  fluid  is  within  the  ring,  and  lute 
with  paraffin.  Incubate  for  5  or  6  days  and  then  examine  under  the  microscope. 
To  make  a  permanent  preparation,  after  examining  in  the  living  state  lift  the  slide 
from  the  cell,  dry,  fix  with  a  drop  of  pure  acetic  acid,  wash  and  stain  with  an  aqueous 
solution  of  eosin.  Wash.  Dry.  Mount  in  balsam. 

2.  Cultures. — The  Tricophyta  are  strictly  aerobic.  Growth  occurs  at 
laboratory  temperature  but  is  more  rapid  at  33°-35°  C.  ;  at  the  higher 
temperatures,  however,  the  culture  soon  becomes  old  and  the  morphology 
of  the  parasites  is  altered.  Primary  cultures  are  best  made  at  the  tempera- 
ture of  the  laboratory.  The  Tricophyta  cannot  be  cultivated  on  acid  media. 
They  grow  readily  on  potato  but  best  on  media  containing  sugar  and  a  little 
nitrogenous  matter,  and  particularly  well  on  beer- wort  (180  per  1,000  of 
maltose),  and  the  following  solution  (Sabouraud)  : 

Sabouraud's  proof  medium. 

Crude  maltose  (Chanut),        -  4  grams. 

Granulated  peptone  (Chassaing),   -  0*75  to  1  gram. 

Distilled  water,    -  -         100  grams. 

Sabouraud's  medium  may  be  solidified  by  adding  T5  parts  of  agar  per 
cent. 

[In  artificial  culture  the  ringworm  parasites  are  subject  to  great  morpho- 
logical variation  both  microscopical  and  macroscopical.  So  considerable  are 
these  pleomorphic  changes  that  unless  the  fact  be  borne  in  mind  a  given 
species  may  easily  be  mistaken  for  another  species  of  the  same,  or  even  of  a 
different,  genus.  Once  a  culture  has  undergone  a  morphological  variation 
there  is  no  known  method  by  which  it  can  be  made  to  reassume  its  original 
characteristics.  To  avoid  as  far  as  possible  pleomorphic  changes  taking 
place  in  cultures  Sabouraud  advises  the  use  of  a  medium  containing  no  sugar 
on  which  to  cultivate  stock  cultures.  The  medium  is  of  the  following  com- 
position : 

Sabouraud's  medium  for  stock  cultures. 

Distilled  water,    -  -         100  grams. 

Granulated  peptone  (Chassaing),    -  -         3-5       ,, 

Agar,  T8  grams.] 

The  isolation  of  the  parasites. — A  diseased  hair,  the  contents  of  a  vesicle 
of  tinea  circinata,  or  a  few  drops  of  blood  from  the  site  of  one  of  the  lesions, 
all  afford  suitable  material  from  which  to  isolate  the  ringworm  parasites. 

(i)  Cultures  from  blood. — Cleanse  the  skin  of  the  affected  part  and  after 
lightly  scarifying  it  collect  a  few  drops  of  blood  and  spread  the  material  on 
sloped  tubes  of  Sabouraud's  maltose  agar. 

(ii)  Cultures  from  vesicles. — Adopting  the  necessary  precautions  to  avoid 
contamination,  remove  the  contents  of  a  vesicle  with  a  fine  pipette  or  a 
platinum  loop  and  sow  the  material  on  tubes  of  the  same  medium. 

(iii)  Cultures  from  hairs.— The  parasite  does  not  occur  in  pure  culture  in  the 
lesions  of  ringworm,  but  is  always  mixed  with  five  or  six  other  species  of 


682  THE  PARASITES   OF  RINGWORM 

organisms.  The  following  is  the  method  advised  by  Sabouraud  for  isolating 
the  parasite  of  ringworm  from  a  diseased  hair  : 

Break  off  one  of  the  affected  hairs,  lay  it  on  a  sterile  slide  and,  with  a 
sterile  cutting  needle,  divide  it  into  as  many  pieces  as  possible.  Sow  each 
fragment  on  a  tube  of  agar  (beer- wort  or  Sabouraud's)  containing  a  large 
percentage  of  maltose — a  medium  on  which  the  other  organisms  accompanying 
the  tricophyton  grow  badly.  As  soon  as  growth  is  visible,  as  indicated  by  a 
downy-looking  speck  at  the  site  where  the  fragment  of  hair  was  sown,  sub- 
cultivate  on  to  another  tube  of  the  same  medium  ;  after  sub-cultivating  two 
or  three  times  in  this  way  transfer  a  portion  of  the  growth  from  the  third  or 
fourth  sub-cultivation  when  about  20  days  old  to  a  slice  of  potato,  rubbing  it 
well  over  the  surface  of  the  medium.  In  this  way  single  colonies  are  obtained. 
The  various  cultures  should  be  grown  at  the  temperature  of  the  laboratory. 

Krai's  method  (p.  691)  is  also  useful.  The  technique  is  more  delicate  but  results 
are  obtained  more  quickly  than  by  Sabouraud's  method. 

Plaut  advises  laying  the  hair  on  a  sterile  slide,  covering  it  with  a  cover-glass  and, 
after  fixing  the  angles  of  the  latter  with  a  little  drop  of  wax,  placing  the  preparation 
in  a  moist  chamber.  After  about  a  week  some  of  the  spores  will  have  germinated 
and  produced  a  mycelium  which  can  easily  be  collected  for  sowing  culture  media. 

When  sub -cultivating  it  is  essential  to  pick  up  only  a  small  portion  of  the 
growth,  and  for  this  purpose  it  will  be  found  more  convenient  to  use  a  steel 
needle  than  the  ordinary  platinum  wire. 

3.  Experimental  inoculation. — The  tricophyta  are  pathogenic  for  man  and 
the  lower  animals.  The  results  of  these  experimental  infections  will  be 
described  when  dealing  seriatim  with  the  different  species. 

[4.  Classification  of  the  Tricophyta. — The  parasitic  species  of  the  genus 
tricophyton  are  divided  into  two  large  groups  : 

[1.  Tricophyton  endothrix. 

[2.  Tricophyton  endo-ectothrix  (or  ecto-endothrix). 

[Pathogenically,  the  endothrix  species  are  confined  to  the  inside  of  the 
hair,  while  the  endo-ectothrix  species  grow  both  within  and  around  the 
affected  hair.  The  former  are  human  parasites  and  infection  takes  place 
from  man  to  man.  The  latter  are  invariably  of  animal  origin  ;  man  becomes 
infected  by  contact  with  a  diseased  animal,  and  the  source  of  the  infection  is 
generally  easy  to  trace. 

[The  endo-ectothrix  species  are  sub-divided,  according  to  the  size  of  the 
"  spores  "  of  the  parasite,  into  Ectothrix  microides  and  Ectothrix  megaspores 
and  in  each  of  these  sub -divisions  there  are  varieties  or  species  differing  from 
one  another  in  cultural  characteristics.  The  small-spored  endo-ectothrix 
species  give  either  a  white,  plastery-looking  growth  (T.  gypseum)  on  the 
maltose  test  medium,  or  a  white,  downy  growth  (T.  niveum).  The  large- 
spored  endo-ectothrix  species  give  either  a  velvety  growth  or  a  culture  like 
the  parasite  of  favus. 

[The  table  on  p.  683  modified  from  Guiart  exhibits  these  points  in  tabular 
form.] 

[A.  Endothrix  species.] 
1.  Tricophyton  tonsurans  (Malmsten). 

Syn. — Tricophyton  megalosporum  endothrix  Sabouraud  ;     [T.  crateriforme 

Sabouraud.] 

Tricophyton  tonsurans  grows  inside  the  hair.  The  affected  hair  breaks 
off  very  short  (3-4  mm.  from  the  skin)  is  thicker  than  the  uninfected  hair 
and  has  no  ring  or  collar  encircling  it.  The  hairs  are  very  difficult  to  epilate 
and  are  occasionally  decolourized.  Tricophyton  tonsurans  is  responsible  for 


TRICOPHYTON  TONSURANS 


683 


a  large  percentage  (42  in  France,  [in  London,  according  to  Colcott  Fox,  38]) 
of  the  scalp  Tricophytoses  of  children. 


GENUS. 

v.  _„_,„,„                    MICROSCOPICAL 

VARIETIES.                               APPEARANCE. 

TYPES. 

CLINICAL 
MANIFESTA- 
TIONS. 

Trico- 
phyton. 

Endothrix. 
Human    origin    occurs 
especially  in  children. 
72  per  cent,  of  cases  of 
tricophyton  ringworm. 
10  per  cent,  of  cases  of 
herpes  circinata. 

Resistant    my- 
celium     with 
square  spores. 

T.  tonsurans  vel  crateri- 
forme. 

Hair 
breaks 
off  long. 
42  per 
cent,  of 
cases. 

Fragile    myceli- 
um with  round 
spores. 

T.  sabouraudi  vel  acu- 
minatum. 

Hair 
breaks 
off  short. 
30  per 
cent, 
cases. 

Ectothrix. 
Animal  ori- 
gin occurs 
especially 
in  the 
adult. 
28  per  cent, 
of  cases  of 
trico- 
phyton 
ringworm. 
90  per  cent, 
of  cases  of 
herpes  cir- 
cinata. 

Ectothrix 
microides. 

Fragile  myceli- 
um with  small 
round  spores. 

T.  gypse- 
um  group. 

T.  menta- 

grophytes 

Suppura- 
tive  con- 
ditions 
(Kerion 
and 
Sycosis). 

T.  niveum 
group. 

T.  felineum. 

Ectothrix 
mega- 
spores. 

Fragile  myceli- 
um with  large 
round  spores. 

Velvety 
growth. 

T.  equinum. 
T.  megnini. 

Faviform 
culture. 

T.  verru- 
cosum 

1.  Microscopical  appearance. — After  treating  with  potash  (p.  690)  or  dis- 
sociating in  a  drop  of  acetic  acid  and  mounting  in  glycerin,  the  affected  hair 
will  be  seen  on  examination  under  the  microscope  to  be  filled  with  numerous 
spores    (pseudo-spores    p.  691)  :    it   is  uncommon   to   find   filaments.     The 
parasite  can  be  recognized  by  the  following  characters : 

(a)  The  pseudo-spores  are  arranged  in  chains :    they  measure  5-6/u,  in 
diameter  and  in  shape  are  round  or  cubical  with  blunted  angles. 

(b)  The  whole  of  the  hair  is  infected  with  the  mycelial  spores. 

(c)  The  mycelial  filaments  show  at  the  most  two  bifurcations,  the  "  tarsal  " 
appearance  is  never  seen  (p.  691). 

(d)  The  parasite  is  entirely  within  the  hair. 

(e)  The  mycelium  can  only  be  broken  up  and  then  with  difficulty  in  a  1  in 
40  solution  of  potash  (resistant  mycelium). 

2.  Cultural   characteristics. — Tricophyton  tonsurans  forms   a   continuous 
cream-coloured  felted  mass  on  the  surface  of  the  medium  on  which  it  is 
growing,  with  fine  thread-like  prolongations  radiating  from  the  centre  to 
the  periphery.     On  maltose-agar  the  centre  of  the  growth  is  depressed  in 
the  form  of  a  flat-bottomed  cupule,  the  inner  sides  being  perpendicular  the 
outer  sloping  [T.  crateriforme].     On  beer- wort  agar  (one-half)  it  forms  a 


684 


THE   PARASITES   OF   RINGWORM 


circular  yellow  growth  with  raised  powdery  centre.     On  potato  numerous 
small  yellowish  and  powdery  star-shaped  growths  appear. 

In  cultures  on  media  containing  maltose,  Tricophyton  tonsurans  gives  origin  to  a 
mycelium  with  spore-bearing  hyphse  arranged  in  racemes.  In  ordinary  peptone 
media  the  growth  is  less  luxuriant  and  the  morphology  is  the  same  as  in  human 
lesions. 


FIG.  324. — Tricophyton  from  a  case  of  "conglomerate  folliculitis."  (T. 
megalosporum  ectothrix.)  The  hair  shaft  is  filled  with  spores  in  chains.  (From 
a  photograph  kindly  lent  by  Dr.  H.  G.  Adamson.) 

» 

3.  Experimental  inoculation. — It  is  difficult  to  infect  man  because  the 
cutaneous  secretions  are  acid  in  reaction  and  the  parasite  will  not  grow  in 
an  acid  medium  (Verujsky).  To  ensure  infection  the  patient  should  be  given 
15-20  grams  of  sodium  bicarbonate  to  render  the  perspiration  alkaline. 
Another  means  of  producing  infection  is  to  cauterize  the  skin  with  the 
red-hot  end  of  a  match  which  has  been  extinguished  ;  this  forms  a  small 
vesicle  containing  a  drop  of  serum  which  is  neutral  in  reaction,  and  on  the 
following  day  the  parasite  can  be  inoculated  into  the  vesicle. 

The  infection  of  animals  (guinea-pigs,  rabbits  and  cats)  is  also  somewhat 
difficult.  After  pulling  out  the  hairs  from  a  small  area  of  the  skin  on  the 
back  scarify  the  latter  and  rub  in  the  culture.  The  lesion  heals  spontaneously 
in  5-6  weeks. 

2.  Tricophyton  sabouraudi. 
[Syn. — Tricophyton  acuminatum  Bodin.] 

T.  sabouraudi  (Blanchard)  corresponds  to  the  T.  endothrix  with  "  fragile 
mycelium  "  of  Sabouraud.  It  occurs  only  in  the  hair. 

Tricophyton  sabouraudi  is  the  cause  of  the  alopecia-like  ringworm  of  children 
TColcott  Fox  found  T.  sabouraudi  in  26  per  cent,  of  cases  of  scalp  tricophytosis 
in  children].  The  affected  hair  breaks  off  level  with  the  skin  and  is  very 
difficult  to  epilate.  Microscopically  the  hair  is  crammed  with  rounded  pseudo- 
spores  which  escape  from  the  broken  surface  of  the  hair  "  like  billiard  balls 
out  of  a  bag  "  (Sabouraud).  The  mycelium  is  composed  of  moniliform  cells, 
and  is  easily  dissociated  in  potash  (fragile  mycelium). 


TRICOPHYTON  MENTAGROPHYTES 


685 


The  growth  on  beer-wort  agar  and  on  maltose-agar  assumes  the  form  of 
a  projecting  cone  with  a  wide  base  and  traversed  by  deep  sulci  passing  from 
the  apex  to  the  base  :  the  colonies  are  creamy- white  in  colour  and  tinted  with 
pinkish-grey  or  greyish-brown  circles.  On  potato  the  parasite  grows  as  a 
straight  brown  streak  and  is  covered  with  a  very  fine  light  brown  powder. 

3.  Tricophyton  violaceum. 

[T.  violaceum  occurs  in  15  per  cent,  of  cases  of  tricophytic  ringworm  in 
children  in  London  (Colcott  Fox).  It  is  characterized  by  the  violet  colour  of 
the  culture  when  about  3  weeks  old.  The  culture  is  more  or  less  acuminate.] 

4.  Tricophyton  sulphureum. 

[This  tricophyton  seems  to  be  peculiar  to  England,  for  while  Colcott  Fox 
found  it  in  21  per  cent,  of  cases  of  scalp  ringworm  in  children  due  to  trico- 
phyta  in  London  it  is  rare  in  France,  and  four  cases  seen  by  Sabouraud  were 
imported  from  England.     The  culture  resembles  the  crater-like  form  of 
T.  tonsurans,  and  is  sulphur-yellow  in  colour.] 
"f  J 
[B.  The  Endo-ectothrix  species.] 

1.  Tricophyton  mentagrophytes. 

[Syn. — Tricophyton  gypseum  Bodin  ;   Tricophyton  asteroides  Sabouraud.] 
This  parasite  which  gives  a  white  growth  in  culture,  and  is  described  by 


FIG.    325. — Tricophyton   mentagrophyies   (Endo-ectothrix).     Hair   from    the 
beard.     (After  Sabouraud.) 


686 


THE   PARASITES   OF  RINGWORM 


Sabouraud  as  Tricophyton  ectothrix  or  Tricophyton  pyogenes,  is  of  animal 
origin.  In  the  horse  it  causes  a  suppurating  folliculitis.  In  the  human  adult 
it  produces  sycosis  or  mentagra  and  onychomycosis  :  in  children  it  gives 
rise  to  tinea  kerion. 

Tricophyton  mentagrophytes  is  pyogenic  and  the  lesions  are  accompanied 
by  dermatitis. 

It  is  an  endo-ectothrix  parasite  and  grows  both  within  and  outside  the 
hair,  forming  a  sort  of  collar  around  the  base  and  affecting  the  epidermal 
covering  of  the  skin  more  than  the  hair  itself.  The  infected  hairs  are  broken 
and  somewhat  bent  at  the  free  end  giving  to  the  affected  area  a  rather  charac- 
teristic untidy  appearance. 

1.  Microscopical  appearance.  —  In  order  to  find  the  parasite  the  small 
downy  hairs  at  the  periphery  of  the  affected  area  should  be  examined,  and 
not  the  dead  full-grown  detached  hairs.  Epilate  the  downy  hair  together 
with  the  epidermal  cone  from  which  it  emerges  and  treat  with  potash.  On 
examining  the  preparation  under  the  microscope  it  will  be  seen  that  the 
mycelial  spores  form  a  compact  mass  in  the  epidermal  covering  of  the  hair. 
The  spores  of  T.  mentagrophytes  are,  as  a  rule,  larger  than  those  of  T. 
endotkrix  :  some  may  reach  a  diameter  of  15-1  8/x. 


FIG.  326.— Unstained  preparation  of  large-spored  ringworm  (T.  megalosporum 
endo-ec.tothrix).  The  fungus  is  outside  the  hair.  (From  a  photograph  kindly 
lent  by  Dr.  H.  G.  Adamson.) 

When  the  parasite  cannot  be  found  on  the  hair  the  pus  from  a  vesicle  which 
has  not  yet  opened  should  be  collected  and  a  small  drop  examined  unstained. 
In  the  pus,  a  small  number  of  spores  having  the  same  characters  as  those  in 
the  hair  will  be  seen  ;  by  using  an  Abbe  condenser  a  quantity  of  very  slender 
and  very  short  mycelial  debris  can  be  found  which  would  escape  observation 
when  only  ordinary  light  is  used.  It  is  difficult  to  stain  preparations  satis- 
factorily :  fuchsin  and  eosin  give  the  best  results. 

2.  Cultural  characteristics. — On  beer-wort  agar,  which  is  the  best  medium, 
the  culture  forms  in  the  first  instance  a  fine  white  downy  tuft  which  after 


TRICOPHYTON  MENTAGROPHYTES  687 

increasing  in  size  becomes  umbilicated  in  the  centre  and  is  surrounded  with 
star-like  rays  ;  at  the  end  of  a  week  it  is  covered  with  a  white,  chalky  dust 
and  in  a  fortnight  the  downy  appearance  is  again  seen  in  the  centre. 

On  maltose-agar  a  white  disc  is  formed  downy  in  the  centre,  powdery  and 
godrooned  at  the  margins. 

On  potato;  a  large  white  track  at  first  downy,  later  chalky,  is  produced. 

T.  mentagrophytes  gives  a  white  growth  on  whatever  medium  it  is  grown  : 
this  fact  is  of  importance  since  all  species  of  Tricophyton  which  give  white 
growths  are  pyogenic. 

Tricophyton  mentagrophytes  can  live  as  a  saprophyte  and  grows  readily  on 
garden  mould,  mulberry  leaves,  etc. 

3.  Experimental  inoculation. — Tricophyton  mentagrophytes  is  pathogenic 
for  man  and  guinea-pigs.  Infection  of  the  guinea-pig  is  easy  ;  it  is  only 
necessary  to  pick  up  a  little  of  the  growth  in  the  teeth  of  a  pair  of  pressure 
forceps  and  then  to  pinch  the  animal's  skin  between  the  teeth  of  the  forceps. 
This  method  of  inoculation  gives  rise  to  a  serpiginous  tricophytosis  which 
persists  indefinitely  but  is  unaccompanied  by  suppurative  folliculitis  (Bodin). 

2.  Tricophyton  equinum. 

Tricophyton  equinum  was  isolated  by  Matruchot  and  Dassonville  during  an  epi- 
zootic of  equine  herpes  which  was  contagious  for  man,  horses,  guinea-pigs  and 
rabbits.  T.  equinum  belongs  to  the  endo-ectothrix  sub-division.  On  agar  it  pro- 
duces colonies  which  are  white  on  the  surface,  but  yellow  or  red  in  the  depth.  It 
grows  with  difficulty  on  potato. 

3.  Tricophyton  caninum. 

This  parasite  was  described  by  Matruchot  and  Dassonville  as  occurring  in  ring- 
worm of  dogs.  It  is  an  ectothrix  parasite  with  round,  ovoid  or  elongated  spores. 
On  sugar-agar,  it  gives  a  flocculent  white  growth.  On  potato,  small  golden-yellow 
colonies.  This  species  is  infective  for  dogs  and  guinea-pigs. 

4.  Tricophyton  felineum. 

[Syn. — Tricophyton  niveum  Sabouraud  •    T.  radians  Sabouraud.  ] 
This  parasite  was  found  in  ringworm  in  cats,  and  is  infective  for  man  and  most 
of  the  domestic  animals  :    in  man,  it  produces  tinea  circinata  dysidrosiforme  (Sabou- 
raud).    It  is  an  ectothrix  parasite  and  pyogenic  :   in  cultures  it  resembles  T.  menta- 
grophytes. 

5.  Tricophyton  megnini. 

Tricophyton  megnini  is  the  cause  of  a  severe  tricophytosis  among  the  Gallinaceae. 
It  may  infect  the  human  hair,  in  which  it  grows  in  the  deeper  layers  in  the  form  of 
numerous  large  spores  and  in  the  superficial  layers  as  fine  mycelial  filaments  forming 
a  network  around  the  hair. 

Cultures  grow  very  slowly  and  have  generally  a  ragged-white-disc  appearance 
more  or  less  radiated. 

6.  The  faviform  tricophyta. 

These  are  the  parasites  (described  by  Bodin)  which  produce  lesions  with  distinct 
ringworm  characteristics,  but  in  cultures  behave  like  parasites  of  the  genus  Achorion  ; 
they  form  indeed  an  intermediate  group  between  the  two  genera.  T.  faviforme  of 
the  ass  and  of  the  horse  are  transmissible  to  man. 

[7.  Tricophyton  concentricum. 
[Syn.— Tricophyton  mansoni  Castellani :   Endodermophyton  concentricum 

Blanchard. 

[It  is  uncertain  whether  tinea  imbricata  (p.  700)  is  due  to  infection  with  a 
species  of  Aspergillus  or  whether  it  is  a  true  ringworm  disease.  According 
to  Manson  the  parasite  causing  the  disease  is  a  tricophyton. 


688  THE  PARASITES   OF  RINGWORM 

[This  uncertainty  arises  because  it  is  doubtful  whether  the  organs  of  fructification 
seen  by  Tribondeau,  Jeanselme,  and  Wehmer  are  accidental  contaminations  due  to  a 
saprophyte  growing  symbiotically  with  the  true  parasite  of  the  disease  or  whether 
the  filaments  and  fructifications  are  all  part  of  one  and  the  same  parasite.  The 
description  of  the  parasite  as  seen  in  the  lesions  is  given  at  p.  700. 

[Nieuwenhuis  has  cultivated  a  parasite  in  every  way  resembling  a  Tricophyton 
by  sowing  epidermal  scales  freshly  taken  from  the  lesions,  and  with  these  cultures 
has  succeeded  in  reproducing  the  disease  in  man.  Tribondeau  with  cultures  of  his 
Aspergillus  has  infected  himself  with  the  disease. 

[Castellani  has  also  been  able  to  cultivate  the  Endodermophyton  concentricum  and 
an  allied  species  E.  indicum  and  in  both  instances  has  reproduced  the  disease  by 
experimental  inoculation  of  his  cultures. 

[The  aetiology  of  tinea  imbricata  must  therefore  be  considered  undetermined  : 
further  research  is  necessary  to  definitely  solve  the  question  (Brumpt).] 

SECTION  II.— THE   GENUS  EPIDERMOPHYTON. 
Epidermophyton  cruris. 

[Syn. — Epidermophyton  inguinale.  ] 

Ringworm  in  the  groin  [Eczema  marginatum,  Tinea  marginata>  Tinea 
cruris]  is  due  to  a  parasite,  described  by  Sabouraud,  Epidermophyton  cruris,1 
closely  resembling  the  other  ringworm  parasites  in  many  of  its  characteristics, 
but  differing  from  them  in  that  it  always  remains  limited  to  the  stratum 
corneum  of  the  epidermis  and  never  attacks  the  hair. 

Epidermophyton  cruris  forms  a  network  of  mycelium  filaments  composed 
of  quadrangular  cells  arranged  end  to  end  growing  horizontally  between  the 
cells  of  the  stratum  corneum. 

The  parasite  grows  on  Sabouraud's  medium  but  produces  no  racemes  of 
spores.  Attempts  to  experimentally  infect  man  and  the  lower  animals 
have  not  been  successful. 


[SECTION  III.— THE   GENUS  MICROSPORUM.] 
1.  Microsporum  audouini. 

Microsporum  audouini  was  first  seen  by  Griiby,  who  found  it  in  an  anomalous 
parasitic  disease  of  the  hair  which  he  called  prurigo  decalvans  (bald  ringworm), 
and  which  was  subsequently  confused  with  alopecia  and  tricophytosis. 

Sabouraud,  however,  cleared  up  the  difficulty  and  showed  that  the  disease  which 
he  calls  teigne  tondante  rebelle  or  teigne  tondante  of  Griiby — bald  ringworm — [and 
which  is  characterized  by  the  presence  of  smooth  bare  spots  of  greater  or  less  extent] 
is  caused  by  infection  with  a  microsporum.  The  parasite  is  properly  designated  Micro- 
sporum audouini,  and  not  Tricophyton  microsporum  as  Sabouraud  originally  described 
it,  since  it  differs  from  the  Tricophyta  both  in  its  appearance  in  the  affected  hairs 
and  in  its  mode  of  growth  in  artificial  culture  (Bodin). 

[Microsporum  audouini  is  the  cause  of  90  per  cent,  of  juvenile  ringworm 
in  London  (Malcolm  Morris).]  It  does  not  grow  on  the  glabrous  skin,  but 
only  on  the  hair.  Hairs  infected  with  M.  audouini  have  a  characteristic 
appearance  :  they  break  off  6  or  7  mm.  from  the  skin,  have  lost  their  colour, 
are  very  thin,  and  are  covered  with  the  parasite  which  imparts  to  them  a 
smooth,  grey  appearance  as  though  they  were  sprinkled  with  a  blue  dust. 

Bodin  has  described  two  varieties  of  Microsporum  audouini. 

M.  audouini  var.  cam's  is* the  cause  of  a  ringworm  in  the  dog ;  it  is  transmissible 
to  man  and  produces  a  condition  similar  to  the  bald  ringworm  of  Griiby ;  inocu- 
lated on  the  guinea-pig  it  leads  to  a  ringworm  which  resolves  spontaneously  in  a 
few  weeks. 

t1  E.  cruris  is  the  cause,  in  some  cases,  at  least,  of  Dhobie  itch.  ] 


MICROSPORUM  AUDOUINI 


689 


M.  audouini  var.  equinum  is  the  cause  of  the  contagious  herpes  of  colts.  In 
cultures  it  is  highly  pleomorphic.  It  is  infective  for  horses,  guinea-pigs  and  dogs. 
The  parasite  may  develop  on  man  and  produce  small,  transient,  erythematous 
lesions. 

[Microsporum  felineum  is  parasitic  on  the  cat  and  may  be  transmitted  to  both 
children  and  adults.  It  grows  with  great  rapidity  in  artificial  culture,  and  is  charac- 
terized by  the  flat  disc-like  appearance  of  its  culture  with  a  small  central  raised 
button  marking  the  site  of  inoculation  of  the  medium.  Cats,  dogs,  and  guinea-pigs 
are  easily  infected  experimentally.] 

1.  Microscopical  appearance. — An  infected  hair  after  being  treated  with 
potash  (p.  690)  will  be  seen  to  be  covered  with  a  mosaic  of  "  mycelial 
spores  "  :  these  spores  measure  1-3/z  in  diameter,  are  round  or  polyhedral 
from  mutual  pressure,  irregularly  agglomerated,  never  arranged  in  chains, 
and  possess  a  clear  transparent  envelope  (fig.  327).  The  "  spores  "  never 
penetrate  into  the  interior  of  the  hair. 


FIG.  327. — Microsporum  audouini.    Photograph  of  an  hair  showing  spores  forming 
a  sheath  around  the  shaft.     (Kindly  lent  by  Dr.  H.  G.  Adamson.) 

Microsporum  audouini  grows  on  the  hair  from  above  downwards  :  the 
older  the  lesion  the  more  deeply  does  the  parasite  sink  into  the  part  around 
the  root  of  the  hair. 

2.  Cultural  characteristics. — Lay  an  hair  on  a  sterile  slide,  cut  it  into 
very  short  lengths,  and  sow  each  piece  on  a  separate  tube  of  one  of  the 
media  used  for  the  cultivation  of  the  Tricophytons.  In  the  majority  of  cases 
a  pure  culture  will  be  obtained  in  the  primary  growths. 

Potato.— On  potato  the  growth  of  M.  audouini  is  characteristic.  After 
7-8  days  a  grey  streak  is  visible  which  tends  to  change  its  colour  to  reddish- 
brown  as  time  elapses  and  about  the  tenth  to  the  twelfth  day  small  bouquets 
of  a  scanty  short  down  appear  here  and  there.  The  fungus  retains  its  vitality 
on  potato  for  several  months  ;  under  similar  conditions  the  tricophyton  dies 
in  18  days. 

Bee.r-wort-agar. — After  3-4  days  tufts  of  the  mycelium  strike  out  and 
grow  into  the  substance  of  the  medium,  taking  the  silky  appearance  of 

2x 


690  THE   PARASITES   OF   FAVUS 

poplar  seeds  :  then  from  the  centre  of  the  colony  a  tuft  of  downy  aerial  hyphse 
emerges,  forming  around  the  growth  a  number  of  glabrous  concentric  circles 
which  ultimately  become  slightly  downy.  The  growth  is  white. 

In  these  cultures  the  mycelial  filaments  are  at  first  short,  but  later  elongate, 
producing  a  tangled  mass  and  becoming  swollen  into  club-shaped  swellings.  After 
a  few  days  the  ends  of  the  mycelial  filaments  throw  out  long  filaments  twisted  like 
the  lash  of  a  whip,  on  which  lateral  thickenings  appear  carrying  a  series  of  teeth 
like  the  teeth  of  a  comb.  These  are  abortive  forms  of  branches.  When  the  parasite 
is  grown  on  unsuitable  media  certain  of  the  filaments  with  club-shaped  swellings 
become  isolated  by  a  transverse  constriction ;  their  contents  become  granular  and 
their  walls  thicken  thus  forming  organs  of  resistance  or  chlamydospores.  In  cultures 
on  suitable  media  fructification  by  fusiform  or  cylindrical  conidia  occurs  at  about 
the  end  of  a  week. 

3.  Experimental  inoculation. — Microsporum  audouini  appears  to  be  a 
strictly  human  parasite.  Experimental  infection  of  children,  whose  scalp  is 
the  most  favourable  situation  for  the  growth  of  the  parasite,  is  not  feasible 
on  account  of  the  contagiousness  and  chronic  nature  of  the  disease. 
Generally  speaking,  the  lower  animals  are  immune  to  infection  (Bodin),  but 
in  a  few  cases  the  disease  has  been  reproduced  in  rabbits,  guinea-pigs  and 
horses  (Courmont). 

The  varieties  found  in  the  spontaneous  ringworms  of  the  dog  and  young 
horse  are  infective  for  the  lower  animals. 


SECTION  IV.— THE   GENUS  ACHORION. 
A.  The  parasite  of  favus  in  man. 

Aehorion  schoenleini. 

Schoenlein  showed  that  favus1  is  caused  by  a  fungus2  belonging  to  the 
genus  Aehorion. 

Aehorion  schoenleini  may  infect  any  of  the  epithelial  tissues — the  hair  of  the 
scalp,  the  skin,  the  nails ;  in  one  case  seen  by  Kaposi  and  Kundrat  the  parasite 
had  infected  the  mucous  membrane  of  the  oesophagus,  stomach,  and  intestine.  As 
a  rule  the  infected  hair  projects  from  a  small  cup-like  depression  in  the  centre  of 
the  characteristic  sulphur-yellow  disc  or  scutulum.  The  hair  is  discoloured  almost 
up  to  its  point  of  emergence,  and  does  not  break  in  the  forceps  but  comes  out  entire. 

According  to  Bodin,  Neebe  and  Unna  several  species  of  Aehorion  are  found  in 
human  favus  all  very  closely  related  to  one  another.  Many  observers  on  the  other 
hand  hold  that  there  is  but  one  species. 

Attempts  to  infect  the  lower  animals  have  given  inconstant  results.  Sabrazes 
says  that  he  has  produced  a  pseudo-tuberculous  condition  by  inoculating  a  spore- 
bearing  culture  of  Aehorion  into  the  peritoneal  cavity  of  a  guinea-pig. 

Morphology  and  methods  of  detection. 

1.  Microscopical  appearance. — Immerse  the  hair  in  a  drop  of  40  per  cent, 
potash  on  a  slide  and  cover  with  a  cover-glass.  Heat  carefully  over  the 
pilot  flame  of  a  Bunsen  until  the  potash  solution  just  begins  to  boil,  then 
lay  the  slide  on  a  cold  surface  to  stop  the  process  of  dissociation  and 
examine  at  once  with  a  low  eyepiece  and  dry  lens  ;  the  potash  clears  the 
hair  and  the  parasite  can  be  readily  seen. 

To  make  permanent  preparations  treat  with  potash  as  above  and  then  run  a  little 
drop  of  eosin-glycerin  under  the  cover-glass.  Hairs  which  have  been  boiled  in 

[x  The  disease  is  so  called  from  its  resemblance  to  an  honeycomb  (L.  favus). 
_  [2  This  perhaps  is  not  strictly  true.     Schoenlein  undoubtedly  found  a  fungus  in  associa- 
tion with  favus  but  it  seems  that  Griiby,  who  independently  discovered  the  fungus,  was 
the  first  to  actually  show  the  relation  of  cause  and  effect.] 


ACHORION  SCHCENLEINI 


691 


FIG.  328.— Vertical  section  through  an  hair 
with  a  portion  of  a  favus  cup. 


potash  must  never  be  washed  in  water  because  contact  with  water  would  reduce 
them  to  powder  at  once. 

In  favus-infected  hairs  treated  with  potash  numerous  mycelial  threads 
will  be  seen,  and  in  addition  very  short,  occasionally  rounded,  bodies — pseudo- 
spores,  or  mycelial  spores — which  are 
resistant  forms,  and  not  true  spores  or 
conidia  which  are  only  produced  in  cul- 
tures. 

The  mycelial  filaments  which  are 
arranged  along  the  axis  of  the  hair  are 
delicate  knotted  and  simple,  or  provided  ^ 
with  two  to  four  branches.  The  pseudo- 
spores  are  3-T/*  in  diameter,  rounded  or 
slightly  flattened ;  they  do  not  infiltrate 
the  whole  of  the  hair  but  form  branched 
chains  separated  from  one  another. 

The  parasite  passes  through  the  epithelium 
to  reach  the  dermis  :  it  destroys  the  hair 
papilla  and  causes  the  hair  to  fall  out.  In 
the  neighbourhood  of  the  favus  cup  there  is 
an  hypertrophy  of  the  epithelial  cells  and,  in 
the  midst  of  these,  masses  of  mycelium 
agglutinated  together  with  an  amorphous 
glairy  substance.  To  study  the  parasite  in 
the  scutulum,  crush  up  one  of  the  scabs 
between  two  slides  and  treat  the  powder 
with  caustic  potash  as  described  above.  A 
scutulum  may  be  embedded  in  paraffin,  cut  and  stained  in  gentian- violet  or  Unna's 
polychrome  blue. 

The  parasite  has  the  following  characteristics  in  the  hair  : 

(a)  There  is  no  visible  envelope.     As  a  matter  of  fact  an  envelope  exists 
but  it  is  very  refractile  and  difficult  to  make  out. 

(b)  The  mycelium  has  a  knotted  appearance  and  the  filaments  composing 
it  are  wavy. 

(c)  The  parasite  never  affects  the  whole  of  the  hair. 

(rf)  The  filaments  divide  into  three  or  four  branches  resembling  the  bones 
of  the  human  tarsus  (tarsefavique). 

2.  Cultural  characteristics.  Conditions  of  growth. — Achorion  schoenleini  is 
distinguished  from  the  moulds  and  resembles  the  tricophyta  in  that  it  does 
not  grow  on  acid  media  (Duclaux  and  Verujski) ;  a  degree  of  acidity  exceeding 
O3  gram  of  tartaric  acid  per  litre  is  sufficient  to  completely  arrest  growth. 
To  obtain  a  culture  of  the  parasite  a  medium  rich  in  peptone  must  be  used ; 
most  carbohydrates  are  unsuitable  :  glycerin  (broth  or  agar)  and  mannite  are 
the  best.  The  parasite  is  aerobic.  Growth  begins  at  15°  C.,  the  optimum 
temperature  is  33°  C.  ;  at  38°  C.  growth  ceases. 

The  appearances  presented  by  Achorion  schoenleini  in  culture  are  not  at 
all  characteristic  :  they  vary  even  when  the  same  strain  is  grown  on  the 
same  medium. 

Achorion  schoenleini  is  not  present  in  pure  culture  in  the  lesions  of  favus 
and  in  order  to  isolate  it  in  pure  culture  Krai's  method  should  be  adopted. 
Grind  up  a  little  piece  of  the  scutulum  in  a  sterile  ground-glass  mortar  with  a 
little  sterile  powdered  silicic  acid.  Plate  the  powder  on  gelatin  in  Petri 
dishes,  examine  the  plate  before  incubating  and  mark  the  spots  where  single 
spores  have  been  sown,  then  incubate  the  plate  and  subsequently  pick  off  the 
colonies  which  develop  in  the  situations  marked. 


692  THE  PARASITES   OF  FAVUS 

Characters  of  growth.  Agar.— On  agar,  a  yellow  brown  wrinkled  layer  is 
formed  with  a  depression  in  the  centre  like  the  cup-shaped  depression  on  the 
scalp. 

Sabouraud's  medium  is  better  than  ordinary  agar. 

Granulated  peptone  (Chassaing),    -  2      grams. 

Pure  anhydrous  glycerin,       ...  4          ,, 

Agar, 1*6 

Distilled  water,    -  -         100      c.c. 

Potato. — A  dry,  raised,  mammillated  layer  brownish-grey  or  brown  in 
colour  appears  about  the  fourth  to  the  fifteenth  day.  The  potato  turns 
brown. 

Broth. — Growth  takes  the  form  of  a  large  spreading  colony  floating  on 
the  surface  of  the  medium.  The  colony  has  the  same  appearance  as  a  growth 
on  agar. 

B.  The  parasites  of  favus  in  the  lower  animals. 

The  parasites  found  in  favus  in  some  of  the  lower  animals  are  related  to,  but  are 
not  identical  with,  Achorion  schcenleini. 

1.  Achorion  quinckeanum. — Favus  in  mice  is  caused  by  Achorion  quinckeanum. 
In  the  lesions,  the  parasite  forms  mycelial  filaments  of  varying  length  consisting 
of  rectangular  or  ovoid  cells  :   the  filaments  break  up  into  short  rectangular  bodies 
which  constitute  the  spores.     The  parasite  grows  readily  at  35°  C.  on  media  con- 
taining glucose  or  glycerin.     It  is  pathogenic  to  guinea-pigs  and  mice  and  gives 
rise  to  scutula  when  a  week- old  culture  on  agar  is  inoculated  on  a  lightly  abraded 
area  of  the  skin. 

2.  Achorion  arloingi. — A.  arloingi  was  found  in  a  case  of  a  ringworm-like  disease 
by  D6sir  de  Fortunet.     It  is  pathogenic  to  mice,  rabbits  and  man. 

3.  Oospora  canina. — Favus  in  dogs  is  due  to  a  related  fungus,  Oospora  canina 
(Costantin  and  Sabrazes). 


SECTION  V.— THE   GENUS  LOPHOPHYTON. 
Lophophyton  gallinse. 

Favus  in  fowls  is  caused  by  Lophophyton  (Epidermophyton)  gallincB  (Megnin). 
[This  parasite  can  be  made  to  infect  man  in  which  case  it  does  not  produce 
the  cup  so  characteristic  of  favus  but  large  erythematous  patches.] 


SECTION  VI.— MICRO-ORGANISMS  IN  ALOPECIA  AREATA, 

A  large  number  of  cases  of  alopecia  are  now  acknowledged  not  to  be  of  a  parasitic 
nature.  In  ordinary  alopecia  numerous  observers  including  the  author  have  con- 
sistently failed  to  detect  any  specific  infecting  agent.  Whatever  the  aetiology  of 
alopecia,  and  there  is  probably  more  than  one  cause  (tropho-neurotic  alopecia,  etc.), 
it  must  be  admitted  that  micro-organisms  rarely  play  any  part  in  the  causation  of 
the  disease. 

It  is  possible  that  some  cases  of  pseudo- alopecia  are  due  to  the  coccus  described 
by  Vaillard  and  Vincent. 


SECTION  VII.— THE  BACILLUS  OF  SEBORRHCEA  OLEOSA. 

[The  micro-bacillus  of  the  "peladic  utricle"  in  alopecia  areata.~] 

Sabouraud  described  a  bacillus  as  being  present  in  seborrhoea  oleosa.  The  patho- 
genic role  of  the  organism  is  not  yet  fully  understood,  but  it  does  not  seem  to  play 
the  part  in  the  causation  of  alopecia  which  he  thought  he  was  justified  in  attributing 
to  it. 


SEBORRHOEA   OLEOSA 


693 


Methods  of  examination. — Remove  one  of  the  crusts,  scrape  it  with  the  edge  of 
a  slide  and  prepare  films  with  the  oily  material  scraped  off. 

Wash  the  films  in  ether  to  get  rid  of  the  fatty  substances,  stain  by  Gram's  method, 
or,  more  simply,  with  blue,  fuchsin,  or  carbol-violet.  The  preparations  show 
numerous  very  fine  bacilli  in  pure  culture  (fig.  329). 

In  young  cultures  the  bacillus  is  punctiform  and 
very  like  a  coccus :  in  older  cultures  the  organism 
is  more  obviously  a  bacillus  and  measures  about 
1  x  O'S/* :  the  organism  stains  readily  with  the  basic 
aniline  dyes,  or  the  ordinary  solutions  containing  a 
mordant  can  be  used.  It  stains  by  Gram's  method. 

Cultures. — It  is  difficult  to  raise  a  culture  of  the 
organism  with  material  from  seborrhcea  of  the  scalp 
or  from  a  comedo.  The  bacillus,  like  all  skin 
bacteria,  requires  an  acid  medium :  on  the  follow- 
ing medium  its  cultivation  is  "  almost  easy." 

Peptone,     -         -  20  grams. 

Glycerin,    -  20      „ 

Glacial  acetic  acid,  -                      5  drops. 

Water,        -  1000  grams. 

Agar,  13       „ 

The  agar  is  distributed  into  tubes  and  sloped. 

To  sow  cultures  from  the  tissues,  wash  the  affected  area  of  the  akin  with  ether, 
then  scrape  it  vigorously  with  the  sharp  edge  of  a  sterile  slide  and  sow  the  sebum 
on  the  surface  of  the  medium.  A  large  quantity  of  the  material  should  be  sown 
on  each  tube.  On  a  few  of  the  tubes,  among  a  number  of  casual  denizens,  one  or 
two  pure  colonies  of  the  bacillus  will  be  obtained. 

At  35°  C.  the  colonies  become  visible  about  the  fourth  day  and,  provided  the 
medium  contain  glycerin,  assume  a  rather  characteristic  appearance,  being  brick- 
red  in  colour  and  in  shape  like  a  pointed  cone. 

In  the  original  cultures,  colonies  of  Sabouraud's  bacillus  are  always  accompanied 
by  other  species  of  micro-organisms,  a  white  coccus,  Staphylococcus  cutis  communis, 
Bacillus  asciformis  (Flaschen  bacillen  of  Unna),  etc.  To  obtain  a  pure  culture  of  the 
seborrhcea  bacillus,  Sabouraud  advises  leaving  the  sebum  for  2  months  between 
two  sterile  slides  or  heating  it  for  10  hours  to  65°-67°  C.  before  sowing  cultures. 
By  this  means  the  organisms  accompanying  it  are  killed  but  the  bacillus  itself  is 
not  destroyed. 

Experimental  inoculation. — Attempts  to  reproduce  the  disease  in  animals  invariably 
fail. 


FIG.  329.— Bacillus  of  seborrhcea 
oleosa.  Seborrhceic  exudate.  Carbol- 
thionin.  (Oc.  II,  obj.  ^th,  Reich.) 


CHAPTER   LI. 
PAEASITES  OF  THE  FAMILY  PERISPORACID.E. 

Introduction. — General  methods  of  examination,  cultivation,  etc. 
Section  I. — The  genus  Aspergillus,  p.  695. 

1.  Aspergillus  glaucus,   p.   695.     2.  Aspergillus   repens,   p.   695.     3.  Aspergillus 
malignus,  p.  695.     4.  Aspergillus  fumigatus,  p.  695.     5.  Aspergillus  pictor,  p.  698. 
Section  II. — The  genus  Sterygmatocystis,  p.  699. 

1.  Sterygmatocystis  nidulans,  p.  699.     2.  Sterygmatocystis  nigra,  p.  699. 
Section  III. — The  genus  Penicillum,  p.  700. 

1.  Pfnicillum  glaucum,  p.  700.     2.  Penicillum  minimum,  p.  700. 
Section  IV. — The  parasite  of  Tinea  imbricata,  p.  700. 

THE  family  of  the  Perisporacidse  comprises  numerous  saprophytic  species 
some  of  which  may  become  parasitic.  They  are  characterized  by  their  septate 
mycelium,  their  conidial  apparatus  and  by  their  asci  surrounded  by  a  complete 
perithecium. 

General  methods. 

1.  Microscopical  examination,     (a)  Of  cultures. — Crookshank  recommends 
the  following  method  : — 

Place  a  drop  of  glycerin  on  a  slide  and  a  drop  of  alcohol  on  a  cover-glass, 
introduce  the  fragments  of  the  fungus  into  the  alcohol,  invert  the  cover-glass 
on  to  the  slide  and  heat  the  latter  over  a  small  flame  until  bubbles  just  begin 
to  appear,  allow  to  cool  and  lute  the  edges  of  the  cover-glass  with  paraffin. 
To  make  a  permanent  preparation  of  an  hanging-drop  culture,  after  examining 
it  in  the  fresh  condition  replace  the  drop  of  culture  fluid  with  a  drop  of  acetic 
acid,  blot  up  the  acid  with  a  piece  of  filter  paper,  stain  with  a  1  per  cent, 
solution  of  safranin  or  eosin  and  mount  in  glycerin  (also  p.  676). 

(ft)  Of  infected  tissues,  i.  Films. — Films  of  pus,  sputum,  etc.  should  be 
fixed  in  alcohol  and  stained  with  a  1  per  cent,  solution  of  safranin  or  with 
carbol-thionin. 

ii.  Sections. — Stain  sections  by  the  method  described  at  p.  675. 

The  method  of  Renon  is  useful  for  species  which  do  not  stain  well  by  Gram's 
method.  Stain  for  several  minutes  with  carbol-thionin,  wash  quickly  in 
distilled  water,  then  in  absolute  alcohol,  pass  through  oil  of  cloves  and  xylol 
and  mount  in  balsam. 

Gaucher  and  Sergent  stain  these  parasites  in  Ehrlich's  violet  for  24  hours 
then  in  Gram's  iodine  solution  for  5  minutes,  decolourize  rapidly  in  absolute 
alcohol,  aniline  oil,  wash  in  xylol  and  mount  in  balsam. 

2.  Cultures. — The  Perisporacidse  grow  best  on  media  of  an  acid  reaction 
and  on  media  containing  sugars.     They  are  aerobic  organisms.     The  optimum 


ASPERGILLUS   GLAUCUS  695 

temperature  of  growth  varies  from  15°-37°  C.  according  to  the  species.  The 
best  media  to  use  are  :  diluted  beer-wort,  Raulin's  liquid  medium  (p.  38), 
milk,  gooseberry- juice,  peptone-broth  containing  sugar  and  glycerin,  potato, 
moist  bread,  and  agar  or  gelatin  made  with  beer-wort  or  Raulin's  medium,  etc. 

These  fungi  may  be  isolated  on  plates  of  gelatin  or  agar  made  with  Raulin's 
medium.  The  method  described  for  the  isolation  of  the  Mucoracidse  is  also 
applicable.  Cultures  in  cells  can  be  prepared  as  described  on  p.  675. 

3.  Experimental  inoculation. — The  pathogenicity  of  the  different  species 
varies  considerably  :  the  amount  of  disease  produced  depends  also  upon  the 
number  of  spores  inoculated.  Birds  are  the  most  susceptible  of  all  animals 
to  the  inoculation  of  parasites  of  this  genus,  then  come  rabbits,  guinea-pigs 
and  monkeys.  It  is  best  to  inoculate  the  material  directly  into  a  vein  but 
infection  also  follows  intra-peritoneal,  sub-cutaneous  and  other  forms  of 
inoculation.  The  lesions  differ  according  to  the  species  inoculated  but 
generally  speaking  partake  of  the  nature  of  a  pseudo-tuberculosis. 

SECTION  I.— THE   GENUS  ASPERGILLUS. 

The  genus  Aspergillus  is  characterized  by  non-septate  spore-bearing  hyphse 
swollen  at  the  tip.  The  swollen  end  is  covered  with  short  branches  or  sterig- 
mata  each  terminating  in  a  row  of  conidia  (fig.  330).  The  arrangement  of 
the  conidial  apparatus  resembles  the  inflorescence  of  an  onion. 

1.  Aspergillus  glaucus. 
Syn. — Aspergillus  herhariorum. 
This  fungus  is  very  widely  distributed  in 
nature.  It  is  often  seen  as  green  spots  on 
decomposing  organic  matter.  It  does  not 
appear  to  be  pathogenic  though  some  observers 
think  they  have  found  it  in  birds  :  possibly 
in  those  cases  the  fungus  was  mistaken  for  a 
variety  of  Aspergillus  fumigatus  (Pinoy).  It 
grows  at  low  temperatures,  but  cannot  be 
cultivated  in  the  warm  incubator  (37°  C.).  The 
spores  are  large  (8-15//.  in  diameter). 

2.  Aspergillus  repens. 

This  species  is  very  closely  related  to  the 

foregoing  from  which  it  is  distinguished  mainly  FIQ  3so^Aspergillus  gUucus, 
by  the  smaller  size  of  its  spores  (4r-8fj-  in 
diameter).  It  has  been  found  in  the  wax  which  sometimes  accumulates  in  the 
external  auditory  meatus  (Siebenmann).  It  does  not  appear  to  have  any 
pathogenic  property. 

3.  Aspergillus  malignus. 

This  species  was  found  by  Lindt  in  the  ear  of  a  man.  It  is  pathogenic 
for  rabbits.  Growth  takes  place  at  35°-37°  C. 

The  swelling  on  the  conidial  hyphse  is  pear-shaped  and  not,  as  in  the  two 
preceding  species,  spherical,  and  for  two-thirds  of  its  area  is  covered  by 
undivided  sterigmata  carrying  chains  or  rows  of  conidia  greenish-white  in 
colour  and  measuring  3-4/x  in  diameter. 

4.  Aspergillus  fumigatus. 

Laulanie  has  shown  that  Aspergillus  fumigatus  is  capable  of  producing  a 
condition  of  pseudo-tuberculosis  when  inoculated  experimentally  into  animals. 


696 


ASPERGILLUS  FUMIGATUS 


Cases  of  pseudo-tuberculosis  due  to  an  Aspergillus  have  also  been  recorded 
by  several  observers  as  occurring  in  the  human  subject.  Aspergillus  fumigatus 
has  been  found  in  infections  of  the  ear  and  naso-pharynx,  and  in  cases  of 
keratitis  with  hypopyon  following  wounds  of  the  eye  caused  by  vegetable 
tissues. 

Pigeon-crammers  are  very  subject  to  aspergillary  pseudo-  tuberculosis.  In  pigeons 
there  is  often  a  "  chancre  "  on  the  buccal  mucous  membrane  due  to  an  Aspergillus. 
The  disease  is  also  found  in  hair-  combers  who  use  flour  of  rye  —  which  is  often  infected 
with  spores  of  Aspergillus  —  for  removing  the  grease  from  hair.  In  human  aspergil- 
lary pseudo-tuberculosis  the  fungus  is  often  associated  with  the  tubercle  bacillus. 
A.  fumigatus  has  also  been  found  in  the  lesions  of  pneumo-  mycosis  in  the  horse  and 
cow.  Renon  has  found  that  by  sowing  millet  seeds,  vetch,  oats,  maize,  wheat,  and 
other  varieties  of  corn  on  appropriate  media  cultures  of  various  species  of  Aspergillus 
can  be  obtained  the  commonest  being  A.  fumigatus. 

1.  Experimental  inoculation.  —  Pigeons,  rabbits,  guinea-pigs  and  monkeys, 
are  susceptible  to  infection  with  A.  fumigatus.  Dogs  and  cats,  on  the  other 
hand,  seem  to  be  immune. 

Pigeons  are  the  best  animals  for  purposes  of  experimental  inoculation. 
The  inoculation  of  2-3  c.c.  of  a  culture  on  Raulin's  medium  into  the  axillary 
vein  leads  to  the  death  of  the  pigeon  in  2  or  3  days.  A  dose  of  1  c.c.  produces 
a  disease  which  runs  a  longer  course  ending  in  death  in  about  a  fortnight. 
By  passage  through  pigeons  the  virulence  of  the  parasite  can  be  raised 
(Kotlair). 

When  death  occurs  soon  after  inoculation,  the  naked  eye  lesions  in  the 
pigeon  are  very  scanty  :  tubercles  will  be  found  in  the  liver,  but  the  lungs 
and  spleen  appear  simply  hypersemic.  When  the  disease  is  of  longer  duration, 
numerous  tubercles  can  be  seen  with  the  naked  eye  in  the  internal  organs  : 
these  are  especially  well  marked  in  the  liver  and  may  show  all  the  stages 
of  development  of  a  typical  tuberculosis  —  (miliary  tubercles,  caseous  degene- 
ration and  fibrous  changes). 

Microscopical  examination  too  shows  that  the  lesions  bear  a  close  general 
resemblance  to  those  of  true  tuberculosis  but  in  all  of 
them  a  thick  felting  of  mycelium  and  spores  is  visible. 

2.  Morphology.  Microscopical  appearance.  —  Aspergillus 
fumigatus  consists  of  a  filamentous  mycelium  with  hyphae 
projecting  from  it  at  right  angles,  these  latter  are  swollen 
into  club-shaped  masses  (spore-bearing  hyphse)  carrying 
undivided  sterigmata  into  which  chains  of  conidia  are 
inserted  :  the  conidia  are  rounded,  smooth,  of  a  brown  or 
green  colour  and  readily  dehisce  ;  they  measure  about  3/u, 
in  diameter.  In  sputum,  a  more  or  less  dense  felted  mass 
of  mycelium  is  found. 

Staining  reactions.  —  Aspergillus  fumigatus  stains  well 
with  the  aniline  dyes  and  is  gram-positive  providing  that 
the  staining  is  prolonged. 

Cultural  characteristics.  —  Aspergillus  fumigatus  grows 
best  in  Kaulin's  liquid  or  beer-wort.  It  is  strictly  aerobic 
and  grows  at  all  temperatures  between  22°  and  40°  C. 

In  broth.  —  Growth  is  very  slow  and  scanty.  Flakes 
°^  m.vcenum  are  found  floating  in  the  medium  which 
remains  clear.  It  is  very  uncommon  to  find  spores  under 
these  conditions. 

In  Raulin  s  medium.  —  Growth  is  abundant  :  numerous  flakes  are  seen 
after  incubating  for  15  hours  at  37°  C.  In  the  culture  tangled  masses  of 
filaments  and  very  numerous  fructifications  will  be  seen  on  microscopical 


tion. 


ASPERGILLUS  FUMIGATUS 


697 


examination.     The  surface  of  the  culture  is  at  first  velvety  and  white  then 
bluish-green,  blackish-green  and  finally,  after  5  or  6  days,  blackish-brown. 

On  gelatin. — Very  small  flakes  slowly  make  their  appearance  along  the 
line  of  sowing  :  a  few  spores  may  appear  about  the  fourth  week  :  in  the  end 
there  is  a  very  slight  liquefaction  of  the  medium. 

On  agar. — After  incubating  for  2  days  at  37°  C.  a  white  film  is  seen  along 
the  line  of  sowing  :  little  by  little  the  growth  acquires  a  green  tint  which 
gradually  deepens  in  colour.  (Agar  made  with  Raulin's  liquid  is  the  best.) 

Grijns  recommends  the  following  medium  : 

Water,         -  -         -         -         100        c.c. 

Extract  of  malt,  -  1        gram. 

Saccharose,  2        grams. 

Agar,  1-75  grams. 

On  this  medium  asci  are  formed. 

On  potato. — An  abundant  growth  rapidly  appears  along  the  line  of  sowing 
which  afterwards  becomes  blackish-green  in  colour. 

3.  Detection  and  isolation  of  the  fungus.    A.  Sputum.— In  suspected 
cases  of  Aspergillosis  the  sputum  should 
be  examined  for  the  parasite  both  by 
microscopical   examination   and  by  cul- 
tures. 

Microscopical  examination.  —  Renon 
recommends  the  following  method :  Pre- 
pare films  with  the  green-coloured  part 
of  the  sputum  and  stain  for  10  minutes 
in  an  aqueous  solution  of  safranm.  The 
mycelium  and  spores  are  stained  pale 
orange.  Carbol-thionin  may  also  be  used. 

Cultures. — Pick  up  some  small  frag- 
ments from  the  centre  of  the  sputum  and 
sow  in  tubes  of  Raulin's  medium.  In- 
cubate at  37°  C.  and  after  2  days  the 
mycelium  will  have  formed  a  whitish, 
velvety  layer  on  the  surface  of  the  medium, 
and  this  soon  becomes  covered  with  green 
spores.  On  inoculating  an  emulsion  of  these  spores  into  the  veins  of  a  rabbit 
or  pigeon  a  fatal  pseudo-tuberculous  aspergillosis  is  set  up  in  a  few  days,  and 
if  a  small  piece  of  the  kidney  of  the  experimental  animal  be  sown  in  Raulin's 
fluid  a  pure  culture  of  the  fungus  can  be  obtained. 

B.  Sections. — Harden  the  tissue  in  alcohol,  embed  in  paraffin  and  stain 
either  by  Gram's  method  or  by  the  following  modification  of  Weigert's  method. 

1.  Stain  with  Orth's  picro-carmine. 

2.  Stain  for  20  minutes  in  carbol-gentian-violet. 

3.  Wash  rapidly  in  0'7  per  cent,  normal  saline  solution  and  blot  with  a 
piece  of  filter  paper. 

4-  Treat  for  1  minute  with  Gram's  solution  and  soak  up  the  excess  with 
filter  paper. 

5.  Transfer  for  a  few  moments  to  aniline  oil. 

6.  Replace  the  oil  with  xylol,  blot  up  the  excess  of  fluid.     Mount  in  balsam. 
One  or  other  of  the  methods  described  on  p.  694  may  also  be  used. 

4.  Toxin. — According   to    Kotliar,    Aspergillus  fumigatus    forms    neither 
toxins  nor  immunizing  substances  in  culture  media.     Cecci  and  Besta  have 
however  extracted  from  the  spores  a  toxic  substance  of  unknown  composition 
which  is  unaltered  by  boiling  and  can  be  preserved  in  alcohol.     In  rabbits, 


FIG.   332.— Mycelium  of  Aspergillus  fumi- 
tus  in  the  sputum  of  an  hair-comber.    (After 


698  ASPERGILLUS   PICTOR 

and  especially  in  dogs,  the  toxin  produces  a  disease  characterized  by  tremors 
and  twitching  of  the  muscles  and  by  respiratory  and  circulatory  disturbances 
which  is  fatal  in  a  few  hours  (these  symptoms  are  comparable  with  those  of 
pellagra). 

Cecci  and  Besta  treat  cultures  rich  in  spores  with  90  per  cent,  alcohol  or 
ether  for  1 2  days.  After  evaporating  the  solvent  a  greenish-yellow  substance  of 
syrupy  consistence  is  left  from  which  all  the  toxin  can  be  extracted  with  water. 

Bodin  and  Gautier  have  obtained  a  toxin,  of  unknown  composition,  possibly 
identical  with  that  of  Cecci  and  Besta,  by  growing  Aspergillus  fumigatus  at 
30°  C.  in  a  solution  of  peptone  containing  a  carbohydrate  (glucose,  saccharose, 
maltose  or  dextrin).  Under  these  conditions  the  culture  becomes  toxic  about 
the  twelfth  day.  The  toxin  is  very  resistant  to  heat  and  is  only  destroyed 
after  heating  at  120°  C.  for  half  an  hour.  When  inoculated  into  rabbits,  dogs, 
guinea-pigs,  cats,  or  mice  it  leads  to  tetanic  and  paralytic  convulsions,  and 
if  the  dose  inoculated  be  sufficient  may  cause  death  in  a  few  hours.  It  should 
be  noted  that  while  the  dog  is  immune  to  an  inoculation  of  spores  it  is  highly 
susceptible  to  the  action  of  the  toxin ;  and  on  the  other  hand  the  pigeon, 
while  very  susceptible  to  the  inoculation  of  spores,  is  unaffected  by  six  times 
the  dose  of  toxin  fatal  to  a  rabbit. 

5    Aspergillus  pictor. 
[Syn. — Tricopliyton  pictor.] 

Pinta  (Fr.  Carates)  is  the  word  used  to  describe  certain  chronic  skin  diseases, 
very  common  in  Central  America,  characterized  in  their  early  stages  by  a 
varied  pigmentation  of  the  skin.  Four  varieties  are  recognized,  the  black, 
the  blue,  the  violet  and  the  red. 


FIG.  333. — Scale  from  the  epidermis  of  a  case  of  the  violet  variety  of  Pinta. 
x  450.     (After  Montoya  y  Florez.) 

[JEtiologically  the  several  forms  of  Pinta  would  appear  to  be  due  each  to  a  different 
species  of  fungus,  the  parasites  differing  from  one  another  in  the  character  of  their 
fructifications.  In  the  red  and  blue  varieties,  for  instance,  the  fructification  is 
similar  to  that  of  an  Aspergillus  (A.  pictor  Blanchard)  in  the  black  variety  to  that 
of  a  Penicillum  (provisy.  P.  pictor  Neveu-Lemaire).  In  other  cases  it  is  of  an 
intermediate  type  (Brumpt).] 

In  man  the  parasite  forms  long,  dichotomously-branched  mycelial  fila- 
ments between  the  epithelial  cells.  Some  of  the  branches  end  in  a  pear-shaped 


THE  GENUS  STERYGMATOCYSTIS  699 

fructification  surmounted  by  a  single  row  of  5  to  6  sterigmata  each  carrying 
a  row  of  3  to  15  spores. 

[According  to  Guiart,  it  cannot  yet  be  considered  as  proved  that  the  fungi 
which  were  described  by  Montoya  y  Florez,  are  the  true  parasites  of  Pinta  ;  Darrier 
and  Bodin  have  found  a  Tricophyton  in  more  than  one  case  of  Pinta  in  Paris.  ] 

For  purposes  of  microscopical  examination,  treat  the  scales  with  warm 
40  per  cent,  potash  (p.  690).  To  prepare  stained  preparations,  treat  with 
ether  to  remove  the  fat,  then  with  absolute  alcohol  containing  acetic  acid  for 
5  minutes,  wash  in  absolute  alcohol,  stain  in  a  dilute  solution  of  Unna's 
polychrome  blue  (10  minutes)  or  thionin  (12-24  hours),  wash  in  absolute 
alcohol  again,  then  in  xylol  and  mount  in  balsam. 

These  fungi  grow  readily  on  glycerin-agar,  beer-wort-agar,  Raulin's  medium, 
potato,  etc.  [Guiart  has  been  able  to  cultivate  the  different  varieties  on 
glycerin  media  and  on  media  containing  iron,  copper  or  zinc  sulphate.  ]  Cul- 
tures should  be  sown  as  described  on  p.  694.  The  optimum  temperature 
of  growth  is  from  25°-35°  C. 

The  disease  has  been  reproduced  in  man  by  Uribe.  Rabbits  are  also 
susceptible.  [Guiart  also  has  produced  lesions  typical  of  the  natural  disease 
by  inoculating  his  cultures  into  man  and  the  lower  animals.] 

The  fungi  of  Pinta  have  been  found  in  the  water  of  certain  gold  mines, 
the  bodies  of  insects,  etc. 

[The  disease  is  not  contagious  and  is  said  to  be  possibly  conveyed  by  the  bites  of 
bugs  and  by  mosquitos  of  the  genus  /Simulium.  ] 


SECTION  II.— THE   GENUS  STERYGMATOCYSTIS. 

The  genus  Sterygmatocystis  is  characterized  by  spore-bearing  hyphae 
terminating  in  a  spherical  enlargement  covered  with  primary  sterigmata 
(fig.  334,  A)  which  divide  and  give  origin  to  several  secondary  sterigmata 
carrying  chaplets  of  conidia  (fig.  334,  B  and  C). 

1.  Sterygmatocystis  nidulans. 

Syn. — Aspergillus  nidulans. 

Siebenmann  attributed  two  cases  of  otomycosis  to  this  fungus.     It  is 
pathogenic  to  animals  (Eidam,  Pinoy)  and  grows  at  all  temperatures  between 
15°-38°  C.     On  culture  media  it  forms  a  chrome- 
green    layer  :    the  enlargements  of  the  conidia- 
bearing  hyphse  are  triangular  with  rounded  edges. 
The  conidia  measure  about  3/x  in  diameter.     This 
parasite  is  the  infecting  agent  in  some  cases  of 
mycetoma  (p.  665). 


2.  Sterygmatocystis  nigra. 

Syn. — Aspergillus  niger. 

This  fungus  is  frequently  found  in  the  form  of 
black  spots  on  decomposing  organic  matter. 
Although  it  has  been  found  several  times  in 
cases  of  otitis  and  various  other  diseases  of  man 
and  animals  it  does  not  seem  to  be  pathogenic. 
It  does  not  grow  at  temperatures  above  25°  C.  FlG-  w*-— Sterygmatocystis 
The  conidial  swellings  have  primary  and  secondary 

sterigmata  carrying  rows  of  black  conidia  measuring  about  4/x  in  diameter 
(fig.  334). 


700 


THE   PARASITE   OF  TINEA  IMBRICATA 


SECTION  III.— THE   GENUS   PENICILLUM. 

Fungi  of  the  genus  Penicillum  possess  septate  conidial  hyphse  dividing  into 
verticillate  or  whorled  branches,  each  carrying  a  cluster  of  spherical  conidia 
ending  at  the  same  height  and  having  the  appearance  of  an  hair  pencil. 

1.  Penicillum  glaucum  (vel  crustaceum)  is  one  of 
the  commonest  moulds  :  it  forms  green  spots  when 
grown  on  bread  and  potato,  and  is  used   in   the 
manufacture  of  Roquefort  cheese.     In   two  cases 
of  chronic  middle-ear  disease,  Maggiora  and  Gra- 
denigo  found  this  fungus  in  the  Eustachian  tube 
mixed  with  various  other  organisms.     It  is  patho- 
genic for  dogs,  rabbits  and  lambs. 

2.  P.  minimum,  a  related  species,  has  been  found 
in  a  case  of  acute  otitis  (Siebenmann). 


SECTION  IV.— THE  PARASITE   OF  TINEA 
IMBRICATA. 

Syn. — Aspergillus  lepidophyton  Wehmer.  [As- 
pergillus concentricus  R.  Blanchard.]  Lepidophyton 
concentricum  Tribondeau. 

Tinea  imbricata,  or  Tokelau,  is  a  disease  of  the 
skin  especially  prevalent   in    Oceania  and  charac- 
terized by  large  epidermal  scales  arranged  in  closely 
set  concentric  rings. 
Tinea  imbricata  is  said  to  be  caused  by  a  fungus  belonging  either  to  the 
genus  Aspergillus  (Wehmer)  or  to  a  very  closely  related  genus,  Lepidophyton 
(Tribondeau). 

[The  relationship  of  Tribondeau' s  parasite  to  tinea  imbricata  is  not  yet  established. 
It  is  quite  possible  that  this  is  an  harmless  saprophyte  and  that  the  true  parasite 
of  the  disease  is  a  Tricophyton — T.  concentricum  Blanchard- — (Brumpt).  ] 

The  parasite  is  found  in  considerable  amount  in  the  epidermal  scales  in 
which  it  forms  septate  and  branched  mycelial  filaments  not  unlike  the  fila- 
ments of  certain  of  the  "  resistant "  Tricophyta.  Some  of  the  filaments 
consist  of  a  series  of  segments  in  the  form  of  grains  of  oats,  which  occasionally 
show  organs  of  reproduction  ending  in  club-shaped  swellings  with  short 
chains  of  spores. 

To  prepare  microscopical  preparations  treat  the  scales  with  alcohol- ether 
to  remove  the  fatty  substances,  then  with  a  4  per  cent,  solution  of  potash 
for  two  minutes,  wash  in  water  and  mount  in  glycerin.  To  stain  permanent 
preparations,  treat  the  scales  after  washing  in  water  with  absolute  alcohol 
tinted  with  eosin,  clear  in  clove  oil,  wash  in  xylol  and  mount  in  balsam. 

[Tribondeau  has  grown  the  parasite  in  pure  culture  on  cocoa-nut  and  on 
banana  and  has  reproduced  the  disease  on  himself  (vide  T.  concent 'ricum, 
p.  687).] 


FIG.  335. — Penicillum  glaucum. 
(After  Schenck.) 


CHAPTEE  LIL 

PARASITES  OF  THE  FAMILY  SACCHARO- 
MYCETIDJE. 

Introduction. 

Section  I. — The  genus  Endomyces,  p.  702. 

Endomyces  albicans,  p.  702. 
Section  II. — The  genus  Saccharomyces,  p.  704. 

1.  Saccharomyces  tumefaciens,  p.  704.     2.  Other  species  of  Saccharomyces.,  p.  705. 
Section  III. — The  genus  Cryptococcus,  p.  706. 
Section  IV. — The  Saccharomycetidse  and  Cancer,  p.  707. 

THE  Saccharomycetidse,  or  yeasts,  are  unicellular  fungi  which  multiply  by 
budding  and  in  which  naked  asci  are  formed  freely  on  the  mycelium.  Numer- 
ous pathogenic  yeasts  have  been  described,  the  chief  of  which  will  now  be 
shortly  dealt  with. 

These  parasites  are  sometimes  described  as  Blastomycetes.1 
Among  the  Blastomycetes  and  with  the  true  yeasts  Guiart  includes  the 
family  of  the  Oididse,  in  which  reproduction  takes  place  as  in  the  yeasts  by 
budding  and  by  asci,  but  which  may  show  at  one  and  the  same  time  both  a 
filamentous  structure  and  a  yeast-like  form.  This  family,  however,  includes 
a  number  of  quite  dissimilar  species  (Chap.  XL VIII.). 

Methods. — The  details  of  technique  for  the  preparation  of  microscopical 
preparations,  and  of  inoculation,  will  be  described  under  each  species.  The 
Saccharomycetidse  grow  on  most  of  the  ordinary  neutral  or  slightly  acid 
laboratory  media,  and  on  vegetable  decoctions,  glycerin-broth,  and  Nsegeli's 
medium  (p.  39)  containing  2  per  cent,  of  glucose.  These  media  solidified 
with  gelatin  or  agar,  as  well  as  glycerin-agar,  potato,  carrot,  etc.  are  all  well 
suited  to  their  growth. 

t1  De  Beurmann  and  Gougerot  propose  the  abolition  of  the  word  Blastomycetes  as  a 
generic  term  on  the  ground  that  it  is  applied  so  loosely  that  one  is  never  sure  of  its  precise 
significance  in  any  given  context.  They  hold  that  the  use  of  the  word  should  be  strictly 
limited  by  its  etymology  (^Xdcrrr)  and  JJ.VKTIS,  budding  fungus),  and  in  that  sense  it 
refers,  as  do  the  words  bacillus,  coccus,  filament,  etc.,  merely  to  a  morphological  appear- 
ance, and  can  lay  no  claim  to  a  generic  grouping. 

[These  observers  classify  the  parasites  dealt  with  in  this  chapter  in  a  family,  Exoascidse, 
which  they  divide  into  three  genera : — Saccharomyces,  Zymonema  and  Endomyces — to 
which  they  provisionally  add  a  fourth,  Cryptococcus,  to  include  various  other  similar 
but  imperfectly  known  parasites  until  such  time  as  further  investigations  shall  have  shown 
to  which  of  the  other  genera  they  properly  belong.  ] 


702  THE   PARASITIC   YEASTS 

SECTION  I.— THE   GENUS   ENDOMYCES. 
Endomyces  albicans  (Vuillemin). 

Synonyms. — Saccharomyces  albicans  Audry  :    Oidium  albicans  Robin  : 
Syringospora  robini  Quinquaud. 

The  parasite  of  thrush  was  discovered  by  Ch.  Robin  who  gave  to  it  the 
name  Oidium  albicans.  Audry  examined  the  organism  and  classified  it  with 
the  yeasts.  Guiart  relying  on  the  fact  that  both  filaments  and  yeast-forms 
can  be  seen  places  it  among  the  Oididse.  [Vuillemin,  however,  demonstrated 
the  formation  of  true  spores  within  the  filaments.] 

Endomyces  albicans  is  present  in  the  air  and  is  constantly  passing  into  the 
respiratory  passages,  but  is  only  able  to  live  on  the  mucous  membrane  of  the 
mouth  when  the  salivary  secretion  is  altered  by  some  pre-existing  disease.  Thrush 
may  infect  the  mucous  membrane  of  the  oesophagus  and  stomach,  and  occasionally 
also  the  mucous  lining  of  the  anus  and  vulva.  Under  certain  exceptional  conditions 
the  parasite  enters  the  blood-stream  and  causes  a  generalized  infection  (Virchow, 
etc.).  Cases  of  thrush  have  been  reported  in  colts  and  calves  and  also,  but  rarely, 
in  birds  (Eberth,  Martin). 

1.  Experimental  inoculation. — Endomyces  albicans  produces  merely  a  local 
lesion  when  inoculated  into  the  anterior  chamber  of  the  eye,  into  the  peri- 
toneal cavity,  or  beneath  the  skin  of  a  rabbit.     Inoculation  of  a  pure  culture 
of  the  fungus  into  the  ear- vein  of  a  rabbit  may  lead  to  a  generalized  mycosis 
with  metastases  in  the  internal  organs,  and  may  terminate  fatally. 

In  guinea-pigs,  intra-peritoneal  inoculation  produces  peritonitis  with  the 
formation  of  false  membranes.  Intra-pleural  inoculation  is  followed  by  an 
effusion  of  fluid  into  the  pleural  cavity  and  the  parasite  may  subsequently 
become  generalized. 

2.  Morphology.    Microscopical  appearance.    1 ..  In  the  tissues. — To  demon- 
strate the  parasite  in   cases   of  thrush,  remove   a   portion  of  one  of  the 
characteristic  whitish  curdlike  patches  seen  in  the  mouth,  break  it  up  in  a 
little  water  on  a  slide,  treat  for  a  few  seconds  with  a  strong  solution  of 
Gram's  liquid  (p.  209)  and  cover  with  a  cover-glass  :   when  the  preparation 
is  examined  under  the  microscope  the  yeasts  will  be  seen  stained  brown  by 
the  iodine.     The  material  may  instead  be  broken  up  in  a  drop  of  acetic  acid 
which  clears  the  epithelial  cells  and  renders  the  parasite  more  readily  visible. 
Dried  films  should  be  stained  with  an  aqueous  solution  of  a  basic  aniline  dye, 
which  will  stain  the  protoplasm  of  the  parasitic  cells. 

The  white  spots  of  thrush  consist  of  the  parasite  mixed  with  epithelial  cells. 
The  Endomyces  has  the  appearance  of  long  tubular  septate  and  entangled 
filaments  mixed  up  with  ovoid  or  rounded  corpuscles  having  a  large  nucleolus 
(the  mycelium  and  spores  of  older  writers). 

2.  In  cultures. — The  appearance  of  Endomyces  albicans  depends  upon  the 
nature  of  the  medium  on  which  it  is  growing,  being  absolutely  different  on 
different  media.  [Generally  speaking,  it  may  be  stated  that  the  more  solid 
the  medium  on  which  it  is  grown,  the  greater  the  tendency  to  spore  forma- 
tion :  the  more  liquid  the  medium,  the  greater  the  tendency  to  the  formation 
of  filaments  (Wills).]  In  broth  cultures,  forms  similar  to  those  just  described 
are  found,  namely  long  entangled  filaments  mixed  up  with  numerous  oval 
cells. — In  wine,  long  filaments  alone  are  seen,  no  oval  cells. — In  cultures  on 
solid  media  only  oval  round  or  irregular  cells  occur,  arranged  singly  or  in 
irregular  groups,  each  cell  being  surrounded  by  a  refractile  membrane  which 
does  not  take  the  basic  dyes  :  some  of  the  cells  will  be  seen  to  be  actively 
budding.— In  Njegeli's  medium  the  growth  assumes  peculiar  characters": 
microscopical  examination  shows  rows  or  chains  of  oval  cells  on  the  ends  of 


THE  PARASITE   OF  THRUSH 


703 


which  large  spherical  forms — chlamydospores — appear  (fig.  328).  These  are 
not  true  spores  but  resistant  forms,  which  sown  on  a  suitable  medium  may 
bud  and  give  rise  to  new  filaments. 


FIG.  336. — Thrush  from  the  mouth,    a,  epithelial  cells  :  b,  rounded  corpuscles : 
d,  g,  1,  k,  mycelial  filaments.     (Ch.  Robin.) 

To  study  the  chlamydospores,  the  Endomyces  must  be  examined  in  a  drop 
of  culture  medium  or  glycerin — water  would  cause  them  to  rupture. 

The  true  reproductive  organs  are  the  asci  which  measure  about  4/u,  in  dia- 
meter and  contain  four  ascospores.     The  develop- 
ment of  the  latter  has  not  been  followed,  but  they 
are  found  in  the  mouth,  and  in  cultures  on  carrots. 

2.  Cultural  characteristics.  Conditions  of  growth. 
— Endomyces  albicans  grows  at  all  temperatures 
between  28°  and  39°  C.  and  on  most  media.  It  is 
strictly  aerobic  and  grows  well  on  slightly  acid, 
neutral,  or  moderately  alkaline  media  :  marked 
alkalinity  hinders  growth  and  interferes  with  the 
formation  of  the  mycelium. 

Cultures  can  be  obtained  by  removing  a  little  of 
the  material  from  one  of  the  lesions  and,  after 
blotting  it  between  sterile  blotting  paper,  rubbing 
it  over  a  gelatin  plate.  It  is  better  however  to 
dilute  the  material  in  .a  little  sterile  water  and  to 
use  a  drop  or  two  of  the  emulsion  for  isolating  the 
organism  on  gelatin  plates. 

Endomyces  albicans  cannot  be  grown  in  saliva  (Roux 
and  Linossier).  This  fact  explains  why  thrush  occurs 
chiefly  in  the  first  two  months  of  life — at  a  time  that 
is  when  the  salivary  secretion  is  not  yet  established — 
and  during  the  course  of  diseases  which  are  accompanied 
by  a  diminution  of  the  secretion. 

Culture  media.     Gelatin. — The  appearance  of  the  colonies  on  gelatin  is 
characteristic.     The  growth  is  rapid,  and  a  number  of  very  white,  spherical, 


FIG.  337. — Endomyces  albicans. 
A,  filaments  from  a  patch  of 
thrush  :  B,  terminal  chlamydo- 
spores :  C,  asci  and  ascospores. 
(After  Vuillemin.) 


704  THE   PARASITIC  YEASTS 

pearly  colonies  appear  which  however  never  attain  any  large  size.  The 
medium  is  not  liquefied. 

Agar. — At  37°  C.  on  agar  there  is  a  rapid  growth  of  white  smooth  spreading 
colonies. 

Potato. — Small  raised  colonies,  dirty  white  in  colour  and  occasionally 
spotted  with  black. 

Carrot. — On  slices  of  carrot,  the  fungus  forms  an  abundant  shiny  white 
growth  in  48  hours. 

Broth,  sterile  wine,  and  Nsegeli's  medium. — Small  white  masses,  the  liquid 
itself  remaining  clear. 

3.  Biological  .proper ties. — The  virulence  of  the  organism  in  cultures  is  very 
variable  :  it  is  attenuated  by  growth  on  artificial  media  and  is  increased  by 
passage  through  animals  (Roger  and  Grasset). 

Cultures  contain  toxins  (Charrin  and  Ostrowsky)  but  no  immunizing  sub- 
stances. Rabbits  can  be  immunized  by  inoculating  them  sub-cutaneously 
with  increasing  doses  of  attenuated  living  cultures  or  by  intra-venous  inocula- 
tion of  increasing  doses  of  such  cultures.  The  serum  of  immunized  animals 
exhibits  agglutinating  properties  (Roger). 


SECTION  II.— THE  GENUS  SACCHAROMYCES. 

1.  Saccharomyces  tumefaciens  (Curtis). 

Curtis  isolated  a  parasite,  to  which  he  gave  this  name,  from  a  case  of  myxo- 
matous  tumour  of  the  thigh  in  a  man. 

1.  Experimental  inoculation.— Mice,  rats  and  dogs  are  all  susceptible  to 
infection. 

Following  the  sub-cutaneous  inoculation  of  a  small  number  of  the  parasites 
into  rats  or  mice  a  tumour  similar  to  that  occurring  in  man  is  formed.  The 
tumour  attains  enormous  dimensions  and  the  animal  may  die  after  a  con- 
siderable time,  but  the  fungus  never  passes  into  the  blood-stream.  Occa- 
sionally, neoplasms  form  in  all  the  internal  organs  which  appear  as  though 
sprinkled  with  small  white  dots.  In  the  tumours  the  parasite  is  always  found 
in  pure  culture. 

In  rabbits,  intra-venous  inoculation  has  negative  results,  while  sub-cutaneous 
inoculation  produces  a  small  abscess  which  undergoes  spontaneous  resolution. 

Guinea-pigs  are  practically  immune.  It  is, 
however,  possible  to  infect  them  by  using  a 
strain  the  virulence  of  which  has  been  raised  by 
growing  it  in  collodion  sacs  in  the  peritoneal 
cavities  of  guinea-pigs  (Wlaef).  The  inoculation 
of  such  cultures  beneath  the  skin  produces 
lesions  in  the  skin,  and  occasionally  a  generalized 
infection. 

2.  Microscopical  appearance.—  In  the  tissues 
S.  tumefaciens  is  encapsulated,  but  in  cultures 
the  capsule  is  generally  absent. 

(a)  On   agar   at   37°  C.  after  incubating  for 

.— Saccharomyces  tume-  48  hours  growth  consists  of  round  or  ovoid 
Culture  °n  agar>  (After  cells  from  3-6/x  in  diameter,  surrounded  by  a 
double -walled  membrane  and  containing  one  or 
two  refractile  granules :  in  young  cultures  the  ovoid  cells  are  more  numerous 
than  the  spherical  cells  and  nearly  all  of  them  have  a  small  bud  at  one  end. 
Methyl  violet  6B  stains  the  centres  of  these  cells  deep  violet  and  the  wall 


SACCHAROMYCES  TUMEFACIENS 


705 


red- violet :  the  refractile  granules  are  unstained.  The  fungus  stains  by 
Gram's  method. 

(6)  In  human  and  animal  tissues,  the  parasite  is  much  larger  and  occurs 
as  spheres  16-20//.  in  diameter  each  with  a  wall  about  0'5/x  thick  and  surrounded 
by  an  hyaline  capsule  from  8-10/*  thick :  ovoid  forms  and  cells  in  an  active 
stage  of  budding  are  also  seen.  The  bud  separates  and  the  mother  and 
daughter  cells  are  then  contained  within  the  same  capsule  :  the  young  buds 
are  filled  with  grains  of  chromatin. 

Sections. — For  sections,  Curtis  recommends  the  following  technique  : — 

1.  Stain  for  a  few  minutes  in  Orth's  carmine. 

2.  Stain  for  10  minutes  in  the  following  solution  : 


Saturated  solution  of  methyl-violet  6B  in  absolute  alcohol, 
1  in  10,000  aqueous  solution  of  caustic  potash, 

3.  Decolourize  for  1  minute  in  a  pyrogallol  solution  : 

Pyrogallol, 

Distilled  water, 


1  c.c. 
9  c.c. 


1  gram. 
100  c.c. 


4.  Dehydrate.     Mount  in  balsam. 

3.  Cultural  characteristics. — S.  tumefaciens  is  an  aerobic  organism. 
Growth  takes  place  at  ordinary  temperatures  but  is  best  at  37°  C.  and  on 
neutral  or  slightly  acid  media. 

Agar.— When  the  culture  is  sown  with  material  from  an  animal  tissue, 
white  opaque  punctiform  colonies  appear  after  2  or  3  days  which  ultimately 
coalesce  but  never  form  an  uniform  streak.  After  sub-cultivating  several 
times  on  agar,  growth  is  more  rapid  and  more  abundant,  and  a  shiny  thick 
creamy  growth  is  obtained  from  which  sub-cultures  can  be  sown  as  long  as 
6  months  afterwards. 

Gelatin. — Along  the  line  of  the  stab,  a  small  white  discontinuous  track  of 
growth  appears  composed  of  punctiform  colonies  which  are  more  numerous 
near  the  surface.  The  medium  is  never  liquefied. 

Broth. — The  growth  is  very  poor  and  consists  of  small  white  flocculi  which 
fall  to  the  bottom  of  the  tube  leaving  the  medium  quite  clear. 

Beer-wort. — The  growth  is  more  copious  than  on  broth  which  is  also  the 
case  with  all  media  which  are  acid  to  the  extent  of  0!3-0'5  of  sulphuric  acid 
per  litre. 

Potato. — At  37°  C.  in  48  hours  a  white  dry-looking  streak  appears  which 
subsequently  becomes   brown.     Growth 
is  more  abundant  on  glycerin-potato. 

On  serum. — No  growth. 

2.  Other  species  of  saccharomyces. 

(a)  Saccharomyces  anginse. — Achalme 
and  Troisier  have  recorded  a  case  of  sore 
throat  clinically  resembling  thrush,  but 
due  to  a  yeast  consisting  of  ovoid  globules 
showing  more  or  less  large  buds. 

5.  angince   grows  easily  on   ordinary 
media.     On  solid  media  the  growth   is 
thick  copious  and  pinkish-grey  in  colour. 
Microscopical   examination    of   cultures 
shows  forms  identical  with  those  found 
in    the    tissues,    and    occasionally    [in 

cultures   grown   on   alkaline   peptone   gelatin]    asci    each   containing   four 
ascospores. 

2Y 


FIG.  339. — Saccharomyces  angince.     (After 
Troisier  and  Achalme.) 


706  THE   PARASITIC   YEASTS 

Similar  fungi  have  been  described  under  the  following  conditions. 

((3)  Saccharomyces  granulatus  in  a  tumour  of  the  human  jaw  (Vuillemin  and 
Legrain). 

[(y)  Atelosaccharomyces l  busse-buschki,  de  Beurmann  and  Gougerot]  (Crypto- 
coccus  hominis,  Vuillemin)  found  in  a  case  of  osteo-myelitis  of  the  tibia  and  giving 
rise  to  a  generalized  infection  (Busse). 

(8)  Saccharomyces  ellipsoideus  [vel  roseus]  found  in  cases  of  middle  ear  disease 
by  Maggiora  and  Gradenigo. 

(e)  Saccharomyces  membranogenes  isolated  by  Steinhaus  from  the  false  membrane 
on  the  trachea  in  a  case  of  diphtheria. 

(£)  Saccharomyces  guttulatus  in  the  intestine  of  the  rabbit  (Robin,  Casagrandi 
and  Buscalioni). 


SECTION  III.— THE   GENUS   CRYPTOCOCCUS. 

Vuillemin  classifies  together  in  a  provisional  genus — Cryptococcus — certain 
Blastomycetes  in  which  the  formation  of  asci  has  never  been  observed. 

1.  Cryptococcus  dermatitis  (Gilchrist  and  Stokes). 

[Syn. — Zymonema 2  gilchristi,  de  Beurmann  and  Gougerot :   Cryptococcus 
gilchristi,  Vuillemin  :    Blastomyces  dermatitis,  Gilchrist  and  Stokes. 

[This  parasite  is  the  cause  of  a  very  chronic  dermatitis  known  in  America 
as  oidiomycosis,  dermatitis  coccidioides,  protozoic  dermatitis,  blastomycetic 
dermatitis,  psorospermiasis,  blastomycosis,  coccidioidal  granuloma  and 
saccharomycosis  (I.  H.  Wright).  The  disease  is  apparently  not  uncommon 
in  America  though  rarely  seen  in  Europe. 

[Microscopical  appearance. — In  the  tissues  the  micro-organism  generally 
occurs  as  spherical  cells  surrounded  by  a  thick  membrane  ;  as  a  rule  the 
cells  are  seen  in  pairs  of  unequal  size  the  smaller  having  been  budded  off  from 
the  larger. 

[In  cultures. — Cells  similar  to  those  seen  in  the  lesions  are  found,  in  addition 
to  short  mycelial  filaments. 

[The  parasite  does  not  appear  to  be  pathogenic  when  inoculated  beneath 
the  skin  of  mice,  guinea-pigs  and  dogs.] 

2.  Cryptococcus  (Saccharomyces)  lithogenes  was  found  by  Sanfelice  in  the 
glands  of  a  bovine  animal  suffering  from  carcinoma  of  the  liver.     The  yeast 
consists  of  spherical  bodies  of  variable  size,  enclosed  within  a  refractile  mem- 
brane, and  often  undergoing  calcareous  degeneration  in  the  tissues.     It  grows 
easily  on  ordinary  culture  media  and  is  pathogenic  for  guinea-pigs,  sheep  and 
mice.     In  guinea-pigs,  it  leads  to  a  generalized  infection  characterized  by 
the  formation  of  nodular  swellings  containing  calcareous  concretions. 

3.  Cryptococcus  linguae  pilosee  was  described  by  Lucet  as  occurring  in 
several  cases  of  black  tongue  :   it  grows  best  on  glucose  media  and  is  patho- 
genic for  mice,  but  Lucet  failed  to  reproduce  the  lesions  of  black  tongue 
experimentally.     In  one  case  Gueguen  found  an  Oospora  (Oospora  lingualis) 
in  association  with  the  Cryptococcus. 

4.  Cryptococcus  farcinosus  is  the  cause  of  epizootic  lymphangitis,  a  con- 
tagious disease  affecting  horses,  mules  [and  cattle]  (Rivolta).     [The  disease 
is  indigenous  along  the  Mediterranean  littoral  particularly  in  Italy,  and  has 
been  imported  into  India,  South  Africa  and  England  probably  with  infected 
mules.]     This  yeast,  consisting  of  oval  or  rounded  cells  often  budding  at  one 
end,  grows  with  difficulty  except  on  potato  or  coagulated  glycerin-glucose- 
horse-serum. 

imperfect.  ]  [2  £1^77  yeast,  vrj/j.a  filament.] 


YEASTS   AND   CANCER 


707 


FIG.  340.— Cryptococcus  tokishigei,  in 
the  pus  of  an  abscess,  with  phagocytosis. 
(After  Tokishiga.) 


5.  Cryptococcus    tokishigei. — The    disease   known   as    "Japan   farcy"    is 
caused  by  a  similar  parasite, — Cryptococcus  tokishigei  (Tokishiga). 

6.  Cryptococcus    degenerans,    isolated    by 
Roncali    from    several   cases    of    malignant 
growths,  etc. 


SECTION  IV.— THE  SACCHAROMYCES 
AND  CANCER. 

By  inoculating  a  guinea-pig  under  the  skin 
with  a  species  of  Saccharomyces  (S.  neofor- 
mans)  which  he  found  in  the  juice  of 
fermented  fruits,  Sanfelice  produced  a  myxo- 
matous  tumour  at  the  site  of  inoculation 
which  killed  the  animal  within  a  month. 

This  observation,  coupled  with  his  discovery  of  the  Cryptococcus  (Saccharo- 
myces)  lithogenes  (vide  ante)  in  the  glands  of  an  ox  suffering  from  carcinoma 
of  the  liver,  gave  a  certain  amount  of  impetus  to  a  theory  previously  advanced 
by  Russell  that  malignant  new  growths  are  the  result  of  an  infection  with 
blastomycetic  fungi. 

The  theory  is  however  now  discarded.  In  the  first  place,  it  is  very  difficult 
to  explain  the  intra -cellular  position  of  a  yeast  in  an  epithelial  cell  (Borrel)  ; 
secondly,  it  would  seem  to  be  proved  that  a  culture  of  a  yeast  has  never  been 
obtained  by  sowing  a  non-ulcerated  malignant  growth  (Curtis) ;  thirdly,  the 
inoculation  of  blastomycetic  fungi  into  the  lower  animals  has  never  given 
rise  to  growths  histologically  comparable  to  sarcomata  or  carcinomata  ;  and 
finally  the  serum  of  persons  suffering  from  malignant  growths  is  totally  devoid 
of  agglutinating  properties  for  the  yeasts  found  in  tumours  by  Sanfelice, 
Curtis  and  others  (Brouha). 

[A.  S.  Grunbaum  fed  several  mice  on  a  yeast  isolated  from  a  mammary 
cancer.  The  majority  of  the  animals  were  unaffected  but  two  which  died 
43  and  46  days  respectively  after  being  fed  showed  nodules  in  the  lungs, 
bronchial  glands  and  intestines.  While  the  cell  proliferation  observed  in 
these  nodules  appeared  to  be  quite  distinct  from  that  seen  in  ordinary  inflam- 
mation it  is  not  suggested  that  anything  in  the  nature  of  a  neoplasm  was 
produced.  "  So  far  as  these  experiments  go  they  neither  support  nor  weaken 
any  parasitic  hypothesis  concerning  the  aetiology  of  new  growths,  which 
hypothesis  indeed,  if  no  specific  parasite  be  assigned  as  the  cause,  is  not  an 
unreasonable  or  an  unlikely  supposition."] 


PART  IV. 
THE   PATHOGENIC   SPIBOCILET^. 


CHAPTER  LIII. 
THE  BLOOD  INHABITING  SPIROCH^TES.1 

A.  Human  spirochaetosis. 
Introduction. 

Section  I. — Experimental  inoculation,  p.  713. 
Section  II. — Morphology  and  methods  of  detection,  p.  714. 

1.  Microscopical  appearance  and  staining  reactions,  p.  714.     2.  Cultivation  of  the 
parasites,  p.  715. 
Section  III. — Serum  therapy,  p.  716. 

The  differentiation  of  the  various  human  spirochaetes,  p.  717. 

B.  Spirochaetosis  in  the  lower  animals. 

1.  Spirochceta  anserina,  p.  717.     2.  Spirochceta  marchouxi,  p.  718.     3.  Spirochceta 
theikri,  p.  719. 

A.  HUMAN  SPIROCHAETOSIS. 

It  is  a  matter  of  historical  interest  that  the  parasite  of  human  relapsing 
fever,  a  spirochaete  discovered  by  Obermeyer  in  1868,  was  the  first  micro- 
organism to  be  found  in  a  strictly  human  disease. 

Under  natural  conditions  relapsing  fever  only  affects  man.  The  parasite  is  present 
in  the  blood  during  the  attacks  of  fever  but  can  only  be  found  occasionally  and  in 
small  numbers  during  the  apyrexial  intervals  :  about  the  time  of  the  crisis  the 
spirochsetes  generally  disappear  from  the  blood  of  the  peripheral  circulation  and  can 
then  only  be  found  in  the  spleen  where  they  are  taken  up  by  the  leucocytes.  The 
disease  can  be  readily  reproduced  in  man  by  the  inoculation  of  blood  containing  the 
parasites.  The  spirochsete  has  been  found  to  remain  alive  for  more  than  a  month 
in  the  intestines  of  leeches  which  have  been  fed  upon  infected  blood,  and  the  disease 
seems  to  be  transmitted  naturally  by  certain  blood-sucking  insects. 

Relapsing  fever  was  first  observed  in  certain  parts  of  Europe  :  Russia, 
Turkey.,  Northern  Germany.  The  disease  is  most  common  among  the  dirtier 
members  of  the  population  and  in  Europe  bugs  (Aoanthia  lectularia)  and  lice 
(Pediculus  corporis)  would  seem  to  be  the  agents  of  transmission. 

[Nicolle,  Blaizot  and  Conseille  have  shown  that  in  Algeria  relapsing  fever 
is  transmitted  by  lice  (Pediculus  vestimenti  and  P.  capitis).  The  bite  of  the 
insect  is  innocuous  and  infection  takes  place  by  the  "  contaminative  "  method, 
the  spirocheetes  being  rubbed  into  the  wound  caused  by  the  bite  of  the  insect 
by  the  scratching  induced  as  the  result  of  the  bite.  The  method  of  infection 

1  The  Spirochsstse  were  formerly  grouped  with  the  Vibrios  among  the  Bacteria  but 
since  Schaudinn's  investigations  they  have  been  regarded  as  Protozoa  and  have  been 
classified  with  the  Trypanosomata  (Flagellata).  The  point  is  however  still  sub  judice. 
Caullery  and  Mesnil,  Doflein  and  also  Borrel  consider  that  these  parasites  occupy  an 
intermediate  position  between  the  Bacteria  and  the  Protozoa. 


712  THE   BLOOD-INHABITING   SPIROCH^TES 

therefore  is  the  same  as  was  shown  by  Leishman  and  by  Hindle  to  obtain 
in  the  case  of  S.  duttoni  and  Ornithodoros  moubata.  These  observers  have 
also  been  able  to  demonstrate  that  hereditary  infection  occurs  in  the  body 
louse,  an  important  fact  which  proves  that  this  insect  is  a  true  intermediate 
host  for  the  spirochsete.  Attempts  to  transmit  the  spirochsete  to  monkeys 
by  means  of  the  tick  Ornithodoros  savignyi  obtained  in  Tripoli  completely 
failed.  In  one  experiment  Nuttall  demonstrated  that  A.  lectularia  fed  on  an 
infected  mouse  and  immediately  afterwards  upon  a  healthy  mouse  trans- 
mitted S.  recurrentis  from  the  former  to  the  latter.] 

Relapsing  fever  also  occurs  in  Asia — India,  Persia,  China  and  other  parts — 
and  in  that  continent  the  disease  is  apparently  propagated  either  by  the 
parasites  mentioned  above  or,  as  in  Persia,  by  a  tick — the  bug  of  Mianeh, — 
Argas  persicus. 

In  [West  and]  East  Africa  relapsing  fever  is  known  as  Tick  fever. 
Lafforgue  has  described  a  similar  disease  in  Tunis.  Tick  fever  differs  some- 
what from  the  European  disease  chiefly  in  being  much  more  fatal  and  in 
the  fact  that  the  fever  is  of  shorter  duration  and  the  spirochsetes  are  less 
numerous  in  the  blood  of  the  peripheral  circulation.  The  African  fevers  are 
conveyed  by  a  tick  (Ornithodoros  moubata)  which  only  bites  at  night.  This 
disease  was  described  by  Dutton  and  Todd,  Kudicke,  Koch  and  others. 

[In  the  case  of  S.  duttoni  the  tick  produces  infection  only  as  a  result  of  the 
entrance  of  the  infective  excreta  into  the  wound  produced  by  the  bite  of 
the  insect  (Leishman,  Hindle).] 

In  the  tick  the  spirochsete  multiplies  on  the  surface  of  the  ovary  and  passes 
into  the  egg  and  young  embryo. 

[Leishman  found  that  S.  duttoni  breaks  up  into  minute  masses  of  chromatin 
• — "  coccoid  granules" — in  the  ova  and  tissue-cells  of  the  tick  Ornithodoros 
moubata  and  it  would  appear  that  these  coccoid  granules  again  develop  into 
spirochsetes. 

[Hollers  has  shewn  that  ticks  may  remain  infective  for  as  long  as  1J  years  after 
their  initial  feed  upon  an  infected  animal.  "  Hollers  has,  moreover  established  the 
fact  that  infected  ticks  fed  on  six  successive  clean  animals  may,  following  upon 
each  feed  of  blood,  lay  six  successive  batches  of  infected  eggs,  from  which  issue  young 
ticks  capable  of  transmitting  the  spirochsete  to  experimental  animals.  Even  more 
remarkable  is  the  fact  that  the  ticks  may  remain  infective  to  the  third  generation 
the  ticks  throughout  the  second  generation  having  been  fed  upon  clean  animals."  *] 

Relapsing  fever  also  occurs  in  America  and  especially  in  the  United  States. 

At  the  present  time  opinion  is  in  favour  of  regarding  the  spirochsetes  found 
in  relapsing  fever  in  different  parts  of  the  world  as  belonging  to  different 
species  (Novy  and  Knapp,  [Nuttall],  Frsenkel  and  others)  of  which  the 
following  are  distinguished. 

Spirochceta  recurrentis  (  =obermeyeri),  the  spirochsete  of  European  relapsing 
fever. 

Spirochceta  duttoni,  the  spirochsete  of  [West]  African  relapsing  fever 
(Tick  fever). 

Spirochceta  novyi,  of  American  fever. 

Spirochceta  carteri,  of  Indian  fever,  which  however  seems  to  be  very  closely 
related  to  S.  recurrentis. 

[Spirochceta  rossii  (S.  kochi),  the  parasite  of  East  African  relapsing 
fever.] 

For  the  present  the  various  spirochsetes  are  differentiated  by  biological 
tests  ;  the  differences  in  morphology  and  in  the  diseases  produced  by  experi- 
mental inoculation  being  in  many  cases  very  slight. 

t1  Nuttall,  Harben  Lectures  1908.] 


HUMAN  SPIROCHJETOSIS  713 


SECTION  I.— EXPERIMENTAL  INOCULATION. 

I.  European  relapsing  fever  can  be  transmitted  from  man  to  man  by 
inoculation  (Munch,  Metchnikoff,  etc.). 

Carter  and  Koch  have  further  shown  that  the  apes  of  the  old  world  can  be 
infected  by  inoculating  them  beneath  the  skin  with  the  blood  of  an  infected 
man.  The  relapses  which  are  so  characteristic  of  the  human  disease  are  not 
always  reproduced  in  inoculated  apes. 

The  phenomena  following  the  inoculation  of  spirochaetes  into  monkeys 
have  been  investigated  by  Metchnikoff  and  by  Soudake witch.  The  Cercopi- 
theci  are  particularly  susceptible  to  inoculation. 

During  the  febrile  attack  the  spirochsetes  are  very  numerous  in  the  fluid  part 
of  the  blood.  At  the  time  of  the  crisis  they  disappear  from  the  peripheral  circula- 
tion but  are  present  in  enormous  numbers  in  the  spleen  where  during  the  apyrexial 
interval  they  are  contained  within  the  leucocytes,  and  masses  of  leucocytes  contain- 
ing spirochsetes  can  be  found  forming  small  microscopic  abscesses.  A  single  leucocyte 
may  contain  several  spirochaetes  and  occasionally  accumulations  of  spirochsetes 
arranged  like  the  spokes  of  a  wheel  can  be  observed  around  some  of  the  leucocytes. 
The  parasites  soon  disappear  from  the  interior  of  the  leucocytes  leaving  only  irregular- 
shaped  granules  and  a  little  later  the  leucocytes  resume  their  normal  appearance. 
Some  of  the  phagocytes  however  have  succumbed  in  the  attack  as  can  be  shown  by 
the  fact  that  their  nuclei  are  destroyed  and  will  not  take  up  staining  reagents.  The 
spirochsetes  are  taken  up  by  the  leucocytes  while  the  former  are  living  so  that  if  a 
little  material  from  the  spleen  of  an  animal  killed  during  the  apyrexial  interval, 
when  all  the  parasites  are  intra- cellular,  be  inoculated  into  a  normal  animal  the 
latter  will  become  infected  ;  from  which  it  is  obvious  that  some  of  the  Spirochsetes 
retain  their  virulence  even  after  being  phagocyted,  and  it  is  these  which  in  some 
way  not  yet  understood  are  responsible  for  the  relapses  occurring  in  the  human 
disease  (Metchnikoff,  Bardach). 

Some  experiments  of  Soudakewitch  are  important  as  showing  the  role  of  the  spleen 
in  relapsing  fever.  The  spleens  of  a  number  of  monkeys  were  removed  and  the 
animals  afterwards  inoculated  with  infected  material  with  the  result  that  all  the 
animals  died,  phagocytosis  was  absent  and  the  numbers  of  the  spirochsetes  went 
on  increasing  until  finally  they  exceeded  in  number  the  red  cells  of  the  blood. 

Mice  and  white  rats  can  be  infected  by  the  inoculation  of  blood  containing 
the  Spirochceta  recurrentis.  After  intra-peritoneal  inoculation  the  mouse 
suffers  from  a  typical  attack  of  the  disease  with  two  or  three  relapses  and 
ultimately  recovers.  The  rat  does  not  as  a  rule  suffer  from  relapses — though 
sometimes  a  single  somewhat  delayed  relapse  may  be  observed  (Breinl  and 
Kinghorn) — and  has  recovered  its  normal  health  in  about  40  hours.  Fiille- 
born  and  Mayer,  Uhlenhuth  and  Handel  have  not  been  able  to  infect  mice  and 
rats  with  human  blood  but  only  with  blood  from  an  infected  Cercopithecus. 

Rabbits  and  guinea-pigs  are  almost  immune. 

II.  West  African  relapsing  fever  (Tick  fever).     The  spirochsete  of  West 
African  relapsing  fever,  S.  duttoni,  is  generally  speaking  more  virulent  for 
the  lower  animals  than  is  Spirochceta,  recurrentis  (Breinl  and  Kinghorn).     The 
Cercopitheci  and  Macacus  monkeys  are  very  susceptible.     Mice   and   rats 
often  die  after  being  inoculated  intra-peritoneally  with  infected  human  blood  ; 
they  suffer  from  a  number  of  successive  attacks,  usually  less  and  less  severe, 
over  a  period  which  varies  from  3-45  days.     The  chief  lesion  is  an  hyper- 
trophy of  the  spleen  with  haemorrhages  into  the  organ. 

Rabbits  also  suffer  from  a  severe  attack  of  the  fever  if  inoculated  intra- 
peritoneally  with  a  large  dose  (5  c.c.  or  more)  of  infected  blood. 
Guinea-pigs  seem  to  be  more  highly  immune  than  rabbits. 

Experimental  inoculation  with  filtered  blood. — Novy  and  Knapp  found  that 
porcelain-filtered  blood  from  rats  infected  with  S.  recurrentis  in  which  on 


714  THE  BLOOD-INHABITING  SPIROOELETES 

microscopical  examination  no  spirochsetes  could  be  found  was  infective  for 
rats. 

The  blood  was  diluted  with  10  parts  of  citrated  normal  saline  solution  and  filtered 
through  new  Berkefeld  bougies  under  a  pressure  of  50  pounds. 

Kepeating  these  experiments  with  S.  duttoni,  Breinl  and  Kinghorn  once 
or  twice  produced  a  mild  infection  in  rats. 

SECTION  II.— MORPHOLOGY  AND   METHODS   OF  DETECTION. 
1.  Microscopical  appearance  and  staining  reaction. 

Blood  should  be  obtained  by  pricking  the  finger.  A  number  of  blood 
films  should  be  spread  and  dried  ready  for  staining,  and  a  drop  of  blood 
should  also  be  examined  in  the  fresh  unstained  condition. 

The  examination  of  fresh  blood.— If  a  drop  of  blood  be  taken  during  an 
attack  of  fever  and  examined  fresh  under  the  microscope,  numerous  spirochsetes 
will  be  seen  lying  between  the  red  cells  of  the  blood,  8-10/x  long,  very  slender 
and  pointed  at  their  ends,  each  showing  six  to  fifteen  spirals.  They  are 
highly  motile  and  scattering  the  red  cells  as  they  go  move  in  a  straight  line 
either  in  an  oscillatory  manner  or  with  a  cork-screw-like  motion.  The 
spirochsetes  tend  to  agglutinate  and  form  rosettes  in  the  blood  of  persons 
suffering  from  relapsing  fever  (fig.  342).  Very  long  organisms  are  sometimes 
seen  measuring  perhaps  100/x  from  end  to  end  ;  these  appearances  are  really 
due  to  the  fact  that  several  individuals  have  become  attached  to  one  another 
end  to  end. 


FIG.  341. — Spirochceta  recurrentis.     Blood  film.     (Oc.  2,  obj.  T\th,  Zeiss.) 

It  is  said  that  the  movements  observed  in  spirochsetes  are  due  to  flagella  which 
stain  only  with  difficulty,  and  four  flagella  have  been  described  arranged  in  bunches 
of  two  at  each  end  of  the  parasite.  Zettnow  using  a  modification  of  Borrel's  method 
(vide  S.  marchouxi)  has  described  a  peritrichial  structure  surrounding  the  S. 
duttoni  the  flagella  being  inserted  all  over  the  surface  of  the  spirochsete.  Novy 
and  Knapp  have  not  been  able  to  demonstrate  lateral  flagella  but  describe  a  long 
single  undulating  flagellum  in  S.  recurrentis  attached  to  one  end  of  the  organism  : 
in  Breinl's  opinion  this  is  merely  a  prolongation  of  the  periplast.  [According  to 
Nuttall,  "  authors  who  claim  that  spirochsetes  possess  flagella  have  been  led  into 
error  by  the  study  of  stained  specimens  of  macerated  spirochaetes,  these  having  been 
rendered  quite  abnormal  in  appearance  through  the  partial  stripping  off  of  their 
outer  layer  or  periplast,  the  myonemes  forming  the  pseudo-flagella.  ] 


HUMAN  SPIROCH^TOSIS  715 

The  spirochsetes  never  form  spores  :  reproduction  takes  place  according  to 
some  observers  by  transverse  division  (Metchnikoff,  Bardach)  and  according 
to  others  by  longitudinal  division  (Schaudinn,  [Nuttall]). 

Spirochsetes  can  be  kept  alive  for  several  days  in  hanging  drop  preparations 
but  the  addition  of  normal  saline  solution  destroys  their  motility  and  causes 
them  to  agglutinate  in  a  few  minutes  (Karlinski). 

If  the  blood  be  collected  as  soon  as  they  begin  to  appear  in  it  the  spirochsetes  can 
be  kept  alive  for  40  days  but  if  collected  just  before  the  crisis  when  they  are  about 
to  vanish  from  the  circulation  they  die  in  a  day  or  two  (Novy  and  Knapp). 

Staining  reactions. — The  S.  recurrentis  is  somewhat  difficult  to  stain  and 
special  methods  have  to  be  adopted.  It  is  gram-negative. 

1.  Blood-films. — Blood-films  should  be  dried  in  the  air  or  in  an  oven  at 
60°-70°  C.  but  should  on  no  account  be  passed  through  the  flame.     To  render 
the  parasites  more  conspicuous  the  haemoglobin  may  be  dissolved  out  of  the 
red  cells  before  staining  the  films.     The  best  method  for  staining  is  Giinther's 
but  any  of  the  methods  described  as  suitable  for  staining  the  Treponema 
pallidum  may  be  used. 

Giinther's  method. — Follow  the  instructions  given  on  p.  206  using  Ehrlich's 
aniline  violet  as  the  dye  and  leaving  it  to  act  for  8-10  minutes. 

The  red  cells  are  unstained,  the  white  cells  and  the  parasites  are  stained 
violet. 

By  staining  the  blood-films  not  only  are  the  Spirochsetes  more  easily  seen  but  they 
appear  much  more  numerous  than  in  fresh,  unstained  preparations. 

2.  Sections. — Pieces  of  the  spleen  should  be  fixed  in  absolute  alcohol  and 
embedded  in  paraffin.     The  sections  may  be  stained  by  NikiforofTs  method. 

Nikiforoff's  method. — 1.  Stain  the  sections  for  24—48  hours  at  the  ordinary 
temperature  of  the  laboratory  in  the  following  solution  : 

Saturated  aqueous  solution  of  methylene  blue,  -  10  c.c. 

Distilled  water,    -  10     „ 

1  per  cent,  alcoholic  solution  of  tropeolein,      -  1     ,, 

10  per  cent,  solution  of  potash,      -         -  4  drops. 

2.  Wash  in  distilled  water,  then  in  alcohol-ether. 

3.  Clear  in  clove  oil  and  xylol  and  mount  in  balsam. 

2.  Cultivation  of  the  parasites. 

Most  attempts  to  grow  Spirochsetes  in  artificial  culture  have  failed. 
Norris,  however,  and  Pappenheimer  and  Flourney  by  sowing  a  few  drops 
of  the  blood  of  an  infected  rat  in  citrated  human  or  rat  blood  noticed  a  con- 
siderable multiplication  of  the  Spirochsetes  after  24  hours  but  the  cultures 
could  not  be  maintained  further  than  the  second  generation. 

Levaditi  sowed  S.  duttoni  in  macacus  serum  previously  heated  to  70°  C. 
and  placed  the  culture  in  collodion  sacs  in  the  peritoneal  cavities  of  rabbits  ; 
in  this  way  he  obtained  luxuriant  cultures  of  the  organism.  Adopting 
Levaditi's  technique  but  using  non-coagulated  rat  blood  in  the  sacs  in  the 
early  cultures  Novy  was  able  to  grow  an  Indian  Spirochsete  for  twenty 
generations  :  the  cultures  which  were  always  scanty  were  virulent  for  rats 
and  retained  their  vitality  for  7  days. 

Duval  and  Todd  seem  to  have  been  able  to  obtain  a  certain  amount  of 
growth  through  two  generations  by  sowing  an  American  Spirochsete  in  a 
complicated  culture  medium.  A  broth  was  made  with  skinned  mice  and 
sterilized  :  egg  yolk  and  defibrinated  mouse  blood  was  then  added  and 
allowed  to  macerate  in  sealed  vessels  in  the  incubator  at  37°  C.  for  6  or  8 
weeks.  The  medium  was  then  sown  with  the  blood  of  an  infected  mouse. 


716  THE   BLOOD-INHABITING   SPIROCILETES 

[Noguchi  has  succeeded  in  cultivating  S.  recurrentis,  S.  duttoni,  S.  rossii 
and  S.  nowyi  in  test  tubes. 

Noguchi' s  method. — The  most  satisfactory  of  the  methods  employed  appears 
to  be  the  following.  Place  a  piece  of  fresh  rabbit  kidney  in  a  sterile  test 
tube,  add  a  few  drops  of  citrated  blood  from  the  heart  of  an  infected  mouse 
or  rat  and  then  about  15  c.c.  of  sterile  ascitic  or  hydrocele  fluid.  A  layer 
of  sterile  paraffin  oil  may  be  poured  on  the  surface  to  prevent  evaporation. 
Incubate  at  37°  C.  Growth  reaches  its  maximum  about  the  7th-9th  day. 
Sub-cultures  may  be  sown  with  about  O5  c.c.  of  a  young  (4th-9th  day) 
culture  but  it  is  well  to  add  a  small  quantity  of  normal  blood  (human  or  rat). 
Noguchi  has  sub-cultivated  S.  rossii  twenty-nine  times  during  a  period  of 
6  months.] 


SECTION  III.— SERUM  THERAPY. 

I.  Once  a  man  has  recovered  altogether  from  an  attack  of  relapsing  fever 
he  is  immune  to  further  attack.     It  is  also — as  a  large  number  of  experiments 
have  shown — beyond  dispute  that  after  recovering  from  an  experimental 
infection  animals  are  immune  [to  further  inoculation  of  the  same  species]. 

II.  Gabritchewsky  has  shown  that  after  the  fever  has  subsided  monkey's 
blood  is  bactericidal  in  vitro. 

A  drop  of  blood  containing  numerous  Spirochsetes  was  collected  from  a  patient 
during  the  febrile  period  and  mixed  with  a  drop  of  serum  taken  from  a  monkey 
during  the  apyrexial  period  :  in  from  l^t  hours  the  Spirochsetes  became  non- motile, 
swollen  "  and  in  short  altogether  changed  in  appearance."  When  on  the  other 
hand  the  blood  was  mixed  with  normal  serum  the  Spirochsetes  remained  alive  for 
from  45-160  hours. 

According  to  Metchnikofi,  and  Soudakewitch,  the  altered  parasites  seen  by 
Gabritchewsky  were  merely  artefacts. 

The  agglutination  reaction  with  the  serum  of  persons  who  have  recovered 
or  are  still  suffering  from  relapsing  fever  gives  very  irregular  results. 

III.  By  inoculating  a  monkey  with  the  serum  of  another  monkey  which 

had  passed  the  crisis  Gabritchewsky  was  able  to  induce 
an  early  crisis  (48  hours,  against  72  hours  followed  by 
a  relapse  in  a  control  animal). 

xv    ^05=^^^fc>C<'-"'         Bardach   inoculated  a  monkey — with  a  temperature  of 
f        v^Spsgxf'        /  39°C-  and  Spirochaates  in  its  blood — with  6  c.c.  of  blood 
/   from  another  animal  taken  4  hours  after  the  crisis  and  found 
i  i   i,     \x    v        that  by  the  next  day  the  temperature  was  normal  and  the 
/  I     |      ^jr/-*^     Spirochsetes  had  disappeared  from  the  peripheral  circulation : 
^j-    i      £          the  temperature  remained  normal  for  7  days  then  the  animal 
had  another  attack  of  fever  and  Spirochsetes  re- appeared  in 
its  blood.     It  is  possible  in  this  case  that  as  a  result  of 
FIG.  342. — Rosette  agglu-     the  inoculation  of  the  serum  the  Spirochsetes  were  phago- 

(GtathV?stS!oan  bl°°d'     °yted  but  not  destr°yed'  and  escaping  from  the  leucocytes 
were  the  cause  of  the  relapse. 

IV.  Novy  and  Knapp  demonstrated  that  the  blood  of  spirochsete-infected 
rats  is  both  bactericidal  and  agglutinating. 

The  Spirochsetes  are  agglutinated  spontaneously  in  blood  taken  during  the  time 
the  temperature  is  falling  while  the  blood  of  rats  which  have  recovered  has  agglutinat- 
ing properties.  The  Spirochsetes  are  agglutinated  end  to  end  often  in  long  threads 
(p.  714)  or  in  rosettes  or  sometimes  in  irregular  masses. 

The  serum  of  rats  which  have  recovered  from  an  infection  has  slight  pro- 
phylactic properties  (Novy  and  Knapp,  Carlisle,  Breinl)  :  it  does  not  prevent 
infection  but  prolongs  the  incubation  period.  This  prophylactic  property  is 


SPIROCILETOSIS   IN  THE   LOWER  ANIMALS  717 

very  much  more  marked  (250  times  more  so)  in  the  blood  of  rats  which  have 
been  hyper-immunized  by  the  inoculation  into  the  peritoneal  cavity  every 
other  day  of  O25-1  c.c.  of  blood  rich  in  spirochsetes.  The  blood  of  animals 
treated  in  this  way  has  therapeutic  properties  as  well  (Novy  and  Knapp). 

If  the  serum  of  such  hyper-immunized  rats  be  inoculated  either  before  or  at  the 
same  time  as  the  virus  it  will  in  quantities  of  0*002  c.c.  protect  a  rat  against  an 
inoculation  of  O'l  c.c.  of  spirochsete-infected  blood.  If  inoculated  in  quantities 
of  2  c.c.  into  rats  showing  Spirochsetes  in  their  blood  it  will  cause  all  the  parasites 
to  vanish  once  and  for  all  within  an  hour.  In  those  species  subject  to  relapses  (mice, 
monkeys)  the  serum  will  prevent  the  initial  attack  but  the  subsequent  relapses  come 
on  as  though  the  animals  had  not  been  treated  with  the  serum. 

The  immunity  conferred  by  an  inoculation  of  the  serum  would  seem  to  last 
more  than  2  months  in  rats,  mice  and  monkeys. 

The  differentiation  of  the  various  human  Spirochsetes. 

The  different  Spirochaetes  parasitic  in  the  blood  of  man  can  be  differentiated 
by  means  of  the  serum  reactions.  The  serum  of  a  rat  hyper-immunized 
against  a  given  species  of  Spirochsete  will  agglutinate  that  species  and  will 
cause  it  to  undergo  granular  disintegration  both  in  vitro  and  in  vivo  (cf. 
Pfeiffer's  phenomenon)  :  but  has  no  action  or  at  most  a  very  slight  action  on 
other  species  (Uhlenhuth  and  Handel). 

The  method  of  crossed  immunity  does  not  afford  such  a  sharp  means  of 
differentiation.  There  is  always  an  active  crossed  immunity  between 
S.  recurrentis  and  S.  novyi  :  crossed  immunity  is  less  often  observed  (in 
about  50  per  cent,  of  cases)  between  S.  recurrentis  and  S.  duttoni  and  very 
seldom  between  S.  duttoni  and  S.  novyi  (Uhlenhuth  and  Handel).  Frankel 
whose  results  are  rather  different  has  observed  no  crossed  immunity  between 
S.  rossii,  the  Spirochsete  found  by  Koch  in  German  East  Africa  and  S. 
duttoni  of  West  African  fever. 


B.  SPIROCH^TOSIS  IN  THE  LO  WER  ANIMALS. 
1.  Spirochceta  anserina. 

8.  anserina,  an  organism  similar  to  8.  recurrentis  was  found  by  Sakharoff  at 
Tiflis  in  a  disease  of  geese. 

Morphologically,  8.  anserina  is  very  like  8.  recurrentis  but  is  a  little  shorter, 
thicker  and  less  undulating.  It  cannot  be  grown  on  the  ordinary  laboratory  media. 

In  infected  geese  the  Spirochsetes  are  present  in  the  blood  for  several  days  then 
they  suddenly  disappear  and  the  birds  die  of  toxaemia  after  surviving  the  infection. 
Post  mortem  the  internal  organs  will  be  found  to  have  undergone  fatty  degeneration. 

The  experimental  disease  has  been  carefully  studied  by  Cantacuzene.  Geese, 
ducks,  young  chickens,  sparrows,  and  pigeons  are  all  highly  susceptible :  adult 
fowls  and  rodents  are  immune. 

To  demonstrate  the  Spirochsete  in  sections  of  the  tissues  Cantacuzene  gives  the 
following  technique  : 

1.  Cut  the  tissues  into  very  small  pieces  and  fix  in  Flemming's  solution  for  24 
hours.     Wash  in  water  for  24  hours. 

2.  Embed  in  paraffin  (xylol  method). 

3.  Cut  very  thin  sections,  fix  them  on  the  slides  and  stain  for  24  hours  in  the 
following  solution  : 

Carbol-fuchsin,     -          -  2  parts. 

Neutral  glycerin,  1  part. 

4.  Wash  rapidly  in  water,  blot  up  the  excess  of  water,  dehydrate  in  several  lots 
of  pure  ether  for  4-6  hours  and  mount  in  balsam  dissolved  in  ether. 

In  susceptible  animals  the  Spirochsetes  multiply  in  the  blood  after  inoculation 
and  disappear  from  the  circulation  as  soon  as  the  temperature  falls.  The  Spirochsetes 


718  THE   BLOOD-INHABITING  SPIROCHSETES 

never  perish  in  the  blood  in  vivo  :  they  undergo  no  bacteriolytic  change  and  are 
not  taken  up  by  the  leucocytes.  As  soon  as  the  fever  passes  off  the  Spirochsetes 
are  phagocyted  by  the  mononuclear  phagocytes  in  the  spleen  :  when  phagocyted 
they  lose  their  staining  capacity  and  are  dissolved  en  bloc  without  undergoing  any 
granular  disintegration.  The  birds  die  after  the  parasites  are  phagocyted  the 
defensive  mechanism  being  unable  to  deal  with  the  toxin. 

2.  Spirochaeta  marchouxi. 

Syn.  —  Spirochceta  gallince. 

This  parasite  which  resembles  the  8.  anserina  was  discovered  by  Marchoux  and 
Salimbeni  in  the  blood  of  fowls  suffering  from  a  disease  prevalent  in  Rio  de  Janeiro. 
The  same  disease  has  been  found  among  fowls  in  Tunis,  in  the  Soudan,  in  Senegal, 
[in  India  (Greig),  in  Cairo  (Bitter),  in  South  Australia  (Johnson  and  Nuttall)]. 

Galli-Valerio  considers  that  8.  marchouxi  and  S.  anserina  are  the  same  organism 
but  Brumpt,  Borrel  and  other  observers  from  the  facts  of  crossed  immunity  believe 
that  the  diseases  of  fowls  due  to  infection  with  spirochsetes  are  different  diseases 
due  to  different  though  closely  related  parasites-:  so  that  they  describe  the  Rio  de 
Janeiro  parasite  as  S.  marchouxi  seu  gallinarum,  the  Tunis  parasite  as  8.  nicolei  and 
the  Senegal  parasite  as  S.  neveuxi. 

8.  marchouxi  is  pathogenic  for  fowls,  geese,  ducks,  sparrows  and  turtle  doves. 
Inoculated  into  rabbits  it  gives  rise  to  a  disease  of  short  duration  which  resolves 
spontaneously. 

The  disease  can  be  transmitted  by  the  inoculation  of  infected  blood  and  by  feeding 
experimental  animals  upon  the  dejecta  of  birds  suffering  from  the  disease.  Under 
ordinary  natural  conditions  the  disease  is  spread  by  a  tick  [  —  A  rgas  persicus  (and  in 
laboratory  experiments  by  A.  reflexus  and  Ornithodoros  moubata)  Marchoux  and 

Salimbeni,  Nuttall,  Balfour,  Greig.]  Ticks  may 
harbour  the  parasites  for  5  months  [or  more]. 

•  In  the  external  media  the  Spirochsetes  lose  their 

vitality  in  48  hours. 

^  Morphology.  —  To  obtain  the  best  preparations 

')  Borrel  advises  defibrinating    the    infected   blood 

and  then  centrifuging  it.     The  Spirochsetes  which 


f  -"  will  be  found  in  the  upper  layers  should  then  be 

washed  and  centrifuged  several  times  and  drops 
^         of  the  last  washings  spread  on  slides  mordanted 
^  ;         with  tannate  of  iron  and  stained  with  fuchsin 

§       ;          (P.  isi). 

^         In  these  preparations  long  flagella  will  be  found 

^^j      all  over  the  bodies  of  the  parasites  and  especially 

^^  at  one  end,  the  other  end  being  as  a  rule  non- 

ciliated  ;    the  short  forms  have  only  the  terminal 

FIG.  343.—  Spirochceta  marchouxi.        tuft  of  flagella.     Some  of  the  parasites  seem  to 
Irishman's  stain,     x  1000.  have  a  long  flagellum  at  one  or  at  both  ends  (cf. 

ante). 

Multiplication  takes  place  by  transverse  division.  [According  to  Nuttall 
spirochsetes  divide  by  longitudinal  division.  ] 

Cultures.  —  Borrel  and  Burnet  succeeded  in  obtaining  freely  growing  cultures  but 
were  unable  to  propagate  them  beyond  the  second  generation.  The  medium  used 
was  citrated  fowl  blood  or  serum  obtained  by  defibrinating  and  centrifuging  the 
blood.  Levaditi  was  able  to  grow  the  spirochsetes  in  collodion  sacs  in  the  peritoneal 
cavities  of  guinea-pigs  using  fowl  serum  as  the  culture  medium. 

[By  adopting  the  method  employed  for  the  cultivation  of  the  human 
blood-inhabiting  spirochsetes  (p.  716)  Noguchi  has  been  able  to  grow 
S.  marchouxi  outside  the  body.  ] 

Immunity.  —  A  first  attack  always  confers  immunity.  Animals  may  be  artificially 
immunized  by  inoculating  them  with  blood  containing  spirochsetes  after  it  has  been 
kept  for  2-4  days  or  heated  to  55°  C.  for  10  minutes.  Serum  from  blood  recently 
collected  from  birds  suffering  from  the  disease  filtered  through  a  Berkefeld  bougie 
has  similar  immunizing  properties. 


SPIROCILETOSIS   IN  THE   LOWER  ANIMALS  719 

The  serum  of  animals  which  have  recovered  from  an  attack  of  the  disease  exhibits 
prophylactic  properties  and  has  a  marked  agglutinating  action  on  the  Spirochsetes 
in  vitro. 

3.  Spirochaeta  theileri. 

[S.  theileri  was  discovered  by  Theiler  in  the  blood  of  cattle  about  Pretoria.] 
Laveran  gave  the  first  published  description  of  the  parasite.  This  spirochsota 
measures  8-30/z  long,  shows  a  variable  number  of  spirals  and  has  no  flagella.  Cattle 
infected  with  the  spirochsete  [often]  show  Trypanosom.es  or  Piroplasmata  in  the 
blood  in  addition,  and  the  presence  of  the  Spirochsete  is  accompanied  by  certain 
changes  in  the  blood  as  for  example  nucleated  erythrocytes  and  erythrocytes  con- 
taining basophile  granules. 

The  disease  is  transmitted  by  a  tick  (Theiler).  His  experiments  were  repeated 
in  France:  A  number  of  larvae  of  Boophilus  (Rhipicephalits)  decoloratus  raised 
from  the  eggs  of  a  tick  which  had  been  fed  upon  an  infected  bovine  in  the 
Transvaal  were  sent  by  Theiler  to  Laveran  and  Vallee  who  placed  them  on  a  cow 
at  the  experimental  farm  at  Alfort.  The  spirochaetes  appeared  in  the  blood  of  the 
cow  14  days  later  but  were  only  found  for  4  days  :  4  days  later  the  cow  developed 
acute  Piroplasmosis  (P.  bigeminum)  and  died. 

Spirochsetosis  in  the  horse  (Theiler),  and  in  sheep  (Theiler  and  Ziemann)  which 
also  occur  in  the  Transvaal  are  apparently  due  to  infection  with  S.  theileri  (Dodd) 
[but  their  identity  cannot  be  regarded  as  established  (Nuttall)]. 


CHAPTER  LIV. 
TREPONEMA  PALLIDUM. 

Introduction. 

Section  I. — Experimental  inoculation,  p.  721. 
Experiments  on  immunization,  p.  724. 
Section  II. — Morphology,  p.  725. 

1.  Microscopical  appearance,  p.  725.     2.  Staining  methods,  p.  726. 
Section  III. — Detection  and  identification  of  the  treponema,  p.  732. 

1.  CoUection  of  material,  p.  732.     2.  Methods  of  examination,  p.  733.     3.  Identi- 
fication of  the  organism,  p.  734. 
Section  IV. — Cultivation  experiments,  p.  736. 
Section  V. — Serum  diagnosis,  p.  737. 

The  nature  of  Wassermann's  reaction,  p.  738.     Wassermann's  technique,  p.  739. 
Chemical  methods,  p.  740. 

THE  infecting  agent  in  syphilis  is  a  spirochsete.  Though  the  organism  had 
been  previously  observed  by  Bordet  and  Gengou,  Schaudinn  and  Hoffmann 
in  1905  were  the  first  to  describe  it,  and  to  them  is  due  the  credit  of  definitely 
identifying  it  with  the  disease. 

Nomenclature. — The  name  given  to  the  organism  by  Hoffmann  was  Spirochceta 
pallida.  But  the  spirochsete  of  syphilis  differs  so  considerably  from  the  other 
spirochsetes  (vide  infra)  as  to  constitute  a  definite  genus  and  Vuillemin  suggested 
the  name  Spironema.  This  name  had,  however,  been  already  appropriated  and 
was  abandoned  in  favour  of  Treponema.  Treponema  pallidum  is  now  accepted  by 
all  authors. 

The  relationship  of  the  treponema  pallidum  to  syphilis. — Of  the  specific 
nature  of  the  Treponema  pallidum  there  can  now  be  no  longer  any  doubt. 
Though  it  has  been  cultivated  outside  the  body  [only  very  recently],  the 
pathogenic  role  of  the  treponema  is  adequately  attested  by  the  following  facts  : 
it  has  been  found-  by  observers  in  all  parts  of  the  world  in  the  lesions  of  the 
primary  and  secondary  stages  ;  it  is  constantly  present  in  the  lesions  of  con- 
genital syphilis  ;  it  is  found  in  the  blood  of  persons  suffering  from  syphilis  ; 
and  it  is  never  found  either  in  healthy  persons  or  in  persons  suffering  from 
diseases  other  than  syphilis. 

Distribution  of  the  parasite  in  the  tissues. — 1.  In  man,  the  Treponema  pallidum  is 
present  in  the  hard  chancre  (Hoffmann  and  Schaudinn,  Metchnikoff  and  Roux  and 
others)  and  can  be  found  in  practically  every  case  :  Ravaut  and  Le  Sourd,  for 
instance,  were  able  to  demonstrate  it  in  17  out  of  19  cases.  It  is  also  found  in  the 
enlarged  glands  associated  with  the  chancre  (Hoffmann  and  Schaudinn  and  others). 

2.  The  treponema  occurs  also  in  the  lesions  of  secondary  syphilis  :  it  is  to  be 
found  in  the  mucous  patches  and  papules,  psoriasis  palmaris,  etc.  (Schaudinn  and 
Hoffmann,  Roux  and  Metchnikoff,  and  others)  :  and  in  sections  of  the  rose  spots 


THE   SPIROCILETE   IN  RELATION  TO   SYPHILIS        721 

(Veillon  and  Girard),  but  in  the  rash  the  organisms  are  very  few  in  number  and 
difficult  to  demonstrate. 

The  blood  is  also  infective  at  times  and  in  a  transitory  manner  during  the  secondary 
period,  and  on  inoculation  into  monkeys  will  produce  syphilis  in  about  50  per  cent, 
of  the  animals  inoculated.  The  number  of  treponemata  in  the  blood  is,  however, 
small,  and  this  fact  explains  the  failure  of  many  observers  to  detect  their  presence. 
Nevertheless,  by  adopting  improved  methods  of  observation,  the  organism  has  been 
found  in  the  peripheral  circulation.  The  treponema  may  also  be  found  in  the  fluid 
obtained  by  blistering  a  non-ulcerated  lesion  of  the  skin  (Levaditi  and  Petresco). 

The  organism  can  almost  never  be  found  in  the  internal  organs  :  though  in  one 
case  Schaudinn  and  Hoffmann  were  able  to  demonstrate  it  in  a  stained  specimen 
of  material  obtained  by  puncture  of  the  spleen.  During  the  secondary  period  the 
secretion  of  the  testes  is  sometimes  infective  but  the  treponema  has  never  been 
demonstrated  in  it. 

3.  In  the  lesions  of  tertiary  syphilis  the  treponema  is  present  but  in  small  numbers 
only ;    these  lesions   are,  however,  infective.     Monkeys,  for  instance,  have   been 
infected  with  fragments  of  gummata  and  with  the  blood  of  a  patient  in  the  tertiary 
stage  (Hoffmann).     Many  observers  have  failed  to  find  the  treponema  by  staining 
material  from  tertiary  lesions  ;    the  organism  has  nevertheless  been  found  in  the 
papules  and  in  gummata.     Reuter  demonstrated  the  organism  in  the  walls  of  the 
aorta  of  a  person  affected  with  syphilitic  aortitis. 

4.  The  treponema  is  found  in  largest  numbers  and  in  every  organ  of  the  body  in 
congenital  syphilis  and  in  this  form  of  the  disease  the  blood  is  much  more  frequently 
infected  than  is  the  case  with  syphilis  in  the  adult.     Thus  it  has  been  demonstrated 
in  the  skin  in  pemphigus,  in  the  bones  in  osteo-chondritis,  and  in  the  inguinal 
lymphatic  glands.     In  the  alimentary  system  it  has  been  found  in  the  mucous  mem- 
brane of  the  cheeks  and  pharynx,  in  the  tonsils,  in  the  walls  of  the  stomach  and  in 
the  liver  and  gall-bladder.     In  the  geni  to -urinary  tract  its  presence  has  been  noted 
in  the  bladder  and  in  the  ovary  and  it  may  even  penetrate  into  the  Graafian  follicles 
(Hoffmann,  Levaditi  and  Sauvage).     It  occurs  in  the  ductless  glands,  spleen,  supra- 
renal glands  and  thymus.     It  is  present  also  in  the  lungs.     In  the  nervous  system 
it  has  been  described  in  inflammatory  foci  in  the  meninges,  in  the  brain  and  in 
the  peripheral  nerves. 

Spirochsetes  are  found  in  the  placenta  only  when  the  infant  has  obvious  manifesta- 
tions of  a  syphilitic  infection. 

5.  The  treponema  is  found  in  the  primary  and  secondary  lesions  of  experimentally 
infected  monkeys  (Roux  and  Metchnikoff,  Neisser,  Hoffmann  and  others).     Although 
the  internal  organs  (spleen,  bone  marrow,  lymphatic  glands)  are  infective  (Neisser) 
the  treponema  has  not  hitherto  been  demonstrated  in  them. 

[Now  that  Noguchi  has  succeeded  in  growing  the  Treponema  palhdum  in 
pure  culture  (p.  737)  and  has  been  able  to  produce  typical  syphilitic 
lesions  in  rabbits  by  the  inoculation  of  his  pure  cultures  there  is  evidence 
that  at  any  rate  the  lesions  produced  in  the  rabbit  by  the  inoculation  of 
syphilitic  material  are  due  to  the  treponema  and  not  to  any  adventitious 
organisms  which  may  have  been  inoculated  with  it.] 


SECTION  I.— EXPERIMENTAL   INOCULATION. 

The  experimental  study  of  syphilis  was  for  a  long  time  very  barren  of 
results.  Inoculation  of  man,  despite  its  seriousness,  was  occasionally  per- 
formed and  the  fundamental  basis  of  the  aetiology  of  the  disease  established. 
On  account  of  the  difficulties  and  dangers  attending  the  inoculation  of 
syphilitic  material  into  man,  experiments  were  undertaken  with  the  object 
of  infecting  the  lower  animals.  Experiments  on  the  ordinary  laboratory 
animals  repeatedly  failed  and  resort  was  had  to  monkeys,  but  here  again, 
inoculation  of  the  lower  monkeys  gave  only  inconstant  and  inconclusive 
results. 

Experiments  were  then  carried  out  on  the  anthropoid  apes  by  Roux  and 

2z 


722  THE   SPIROCELETE   OF  SYPHILIS 

Metchnikoff,  who  in  1903  demonstrated  that  these  animals  were  susceptible 
to  the  virus  of  syphilis. 

I.  Chimpanzee. — The  chimpanzee  is  the   most  suitable  animal   for    the 
purpose   of  studying   syphilis   experimentally.     Compared   with   man   this 
species  may  be  regarded  as  equally  susceptible  to  syphilis.     Inoculation 
under  the  conditions  described  below  is  certain  to  result  in  infection  (Roux 
and  Metchnikoff). 

The  virus  is  best  obtained  from  a  chancre  or  secondary  lesion  and  should 
be  inoculated  into  superficial  scratches  made  on  any  part  of  the  body,  but 
preferably  on  the  superciliary  ridges,  upper  eyelids  or  genital  mucous  mem- 
brane. Infection  cannot  be  produced  by  inoculation  beneath  the  skin,  into 
the  peritoneal  cavity  or  into  the  blood  vessels. 

The  incubation  period  averages  31  days  (22-35).  Exceptionally  it  may  be 
reduced  to  15  or  prolonged  to  49  days. 

Chancre. — After  a  period  of  incubation  has  elapsed,  small,  rose-coloured, 
hardly  visible  pimples  make  their  appearance  at  the  site  of  inoculation.  Two 
or  three  days  later  the  lesion  is  redder  and  a  small  scale  forms  on  the  surface 
followed  by  ulceration  and  induration  of  the  subjacent  tissues,  reproducing 
exactly  the  appearance  of  the  chancre  as  seen  in  man.  The  corresponding 
glands  become  enlarged  and  indurated. 

Secondary  manifestations. — Secondary  lesions  have  been  observed  in  66  per 
cent,  of  chimpanzees  inoculated  and  are  recognizable  about  a  month  after 
the  appearance  of  the  chancre. 

The  rash  is  very  difficult  to  detect  because  chimpanzees  suffer  naturally 
from  rashes  on  the  skin  which  may  very  easily  be  mistaken  for  a  typical  rose 
rash  (Roux  and  Metchnikoif).  On  the  other  hand,  papules  and  mucous 
patches  are  seen  and  inoculation  from  these  into  another  chimpanzee  results 
in  infection  of  the  latter.  During  the  secondary  stage,  Roux  and  Metchnikoff 
have  observed  attacks  of  paraplegia  coming  on  a  few  weeks  after  the  appear- 
ance of  the  chancre  but  soon  passing  off.  The  spleen  is  frequently  enlarged. 

One  of  the  chimpanzees  inoculated  by  Roux  and  Metchnikoff  suffered  from 
a  malignant  form  of  syphilis  and  died  about  10  months  after  infection. 

Tertiary  manifestations. — Tertiary  lesions  have  not  up  to  the  present  time 
been  observed  in  chimpanzees. 

II.  Orang-outang. — Inoculation  of  syphilitic  material  into  an  orang-outang 
invariably  produces  a  chancre  (Neisser,  Roux  and  Metchnikoff). 

The  incubation  period  is  shorter  than  in  the  chimpanzee  and  averages 
24  days. 

The  chancre  is  not  so  distinct  as  in  the  chimpanzee.  Secondary  mani- 
festations apparently  never  develop. 

HI.  Gibbon. — The  results  of  the  inoculation  of  the  gibbon  are  almost  the 
same  as  in  the  orang-outang  (Metchnikoff  and  Neisser).  Secondary  mani- 
festations are  uncommon.  Neisser  has  noticed  a  papular  eruption  covering 
the  face,  palms  of  hands,  abdomen,  buttocks  and  mucous  membranes. 

As  has  been  pointed  out  already  the  higher  apes  may  be  infected  by  scratching 
infective  material  taken  from  a  primary  or  secondary  lesion  into  the  skin  of  any 
part  of  the  body.  When  material  from  tertiary  lesions  was  used  five  only  out  of 
seventeen  experiments  were  successful  (Neisser,  Sieber  and  Schacht).  Hoffmann 
inoculated  four  animals  with  the  blood  of  a  syphilitic  man  (taken  40  days  and  6 
months  after  infection)  while  it  was  still  warm  and  before  it  had  coagulated.  Two 
of  the  animals  developed  syphilis.  The  same  observer  succeeded  in  infecting  a 
monkey  with  mucus  from  the  nose  of  a  diseased  person. 

Investigation  shows  that  the  virus  of  syphilis  diffuses  very  rapidly  through  the 
tissues.  Excision  of  the  site  of  inoculation  8  hours  after  infection  does  not  prevent 
the  development  of  a  chancre.  During  the  incubation  period  the  bone- marrow  and 


EXPERIMENTAL  INOCULATION  723 

spleen  may  contain  the  virus  and  inoculation  of  either  of  these  tissues  into  monkeys 
may  result  in  infection. 

IV.  The  lower  monkeys. — The  lower  catarrhine  monkeys  may  be  infected 
with  syphilis  but  the  proportion  of  successful  inoculations  is  very  variable. 
Roux  and  Metchnikoff  taking  the  results  of  their  inoculations  of  macacus  and 
cynocephalus  monkeys  together  (M.  rhesus,  M.  cynomolgus,  M.  sinicus, 
C.  hamadryas)  succeeded  in  infecting  50-60  per  cent.  They  affirm  that  the 
only  chance  of  success  lies  in  inoculating  the  material  on  the  orbital  arches  or 
genital  mucous  membrane.  In  experiments  on  the  same  species  of  monkeys, 
Finger  and  Landsteiner  infected  87  per  cent,  by  making  deep  scarifica- 
tions and  inoculating  a  large  amount  of  the  virus.  Thibierge  and  Ravaut 
say  that  macacus  monkeys  can  always  be  infected  if  inoculated  on  the  free 
margin  of  the  eyelids.  The  incubation  period  is  shorter  than  in  the  anthropoid 
apes  and  averages  23  days.  The  primary  sore  takes  the  form  of  an  ulcer 
and  the  subjacent  tissue  is  infiltrated  but  not  characteristically  indurated. 
In  M .  rhesus  the  chancre,  as  a  rule,  has  the  appearance  of  a  papule.  There 
is  no  enlargement  of  the  glands.  Secondary  lesions  have  not  been  ob- 
served. 

As  a  result  of  their  experimental  inoculations,  Roux  and  Metchnikoff  lay 
down  the  following  law  :  the  shorter  the  incubation  period,  the  less  severe  the 
syphilitic  infection. 

V.  Rabbits. — The  rabbit  can  be  infected  by  inoculating  the  virus  into  the 
eye.  Thus,  material  from  hard  chancres  or  secondary  papules  when  inoculated 
into  the  anterior  chamber  of  the  eye  of  a  rabbit  leads  to  the  development  of 
a  small  swelling  of  the  cornea  in  50  per  cent,  of  cases  about  10  days  after 
inoculation  ;  this  swelling  is  subsequently  accompanied  by  a  parenchymatous 
keratitis  with  a  very  marked  lymphocytosis  (Bertarelli).  Microscopical 
examination  shows  the  presence  of  treponemata  in  very  large  numbers  in 
the  anterior  part  of  the  infected  cornea  but  they  are  never  found  in  the 
epithelial  layer.  If  a  small  portion  of  the  infected  cornea  be  transferred  to 
the  anterior  chamber  of  the  eye  of  a  normal  rabbit  a  similar  lesion  containing 
actively  multiplying  spirochsetes  develops. 

The  rabbit  virus  is  capable  of  infecting  monkeys.  Bertarelli  produced 
very  distinct  chancres  and  syphilitic  keratitis  in  Macacus  cynomolgus  by 
inoculating  the  monkeys  either  on  the  cornea  or  by  scarifying  the  skin  with 
material  from  the  cornea  of  the  fifth  passage  rabbit. 

Bertarelli's  results  have  been  confirmed  by  Sherber,  Greef  and  Clausen 
and  others. 

To  ensure  a  successful  result  Bartarelli  recommends  inoculating  the  infected  tissue, 
or  scraping  into  the  anterior  chamber  of  the  eye.  Alternatively,  the  margin  of  the 
cornea  may  be  scarified  and  rubbed  with  the  virus  ;  it  is  important  that  the  eyelids 
be  held  open  for  a  little  while  after  the  operation.  The  material  should  previously 
to  inoculation  be  well  washed  in  sterile  water  to  remove  any  contamination  that 
may  be  present  on  the  surface. 

VI.  Some  of  the  other  lower  animals  appear  to  be  susceptible  to  an  experi- 
mental infection  with  syphilis. 

The  rabbit  virus  on  inoculation  into  guinea-pigs  produces  keratitis  with 
multiplication  of  the  treponemata  (Bertarelli). 

Dogs  (Hoffmann  and  Bruning),  sheep  (Bertarelli)  and  cats  (Levaditi  and 
Yamanouchi)  can  all  be  infected  by  scarifying  the  cornea. 

Siegel  produced  indurated  nodules  at  the  site  of  inoculation  in  mice  2  days 
old  by  inoculating  them  with  the  virus  of  syphilis.  The  spleen  of  one  of 
these  when  inoculated  into  a  monkey  produced  a  very  distinct  syphilitic 
lesion. 


724  THE   SPIROCILETE   OF   SYPHILIS 

Experiments  on  immunization. 

It  is  a  general  rule  that  an  individual  with  a  chancre  cannot  be  re-infected, 
but  the  rule  is  not  without  exceptions  even  in  the  case  of  the  human  subject 
(Queyrat).  In  monkeys,  a  second  inoculation  10  days  after  the  chancre  has 
appeared  may  give  rise  to  a  second  chancre  (Roux  and  MetchnikofE). 

Individual  human  subjects  exhibit  considerable  variation  in  their  suscepti- 
bility to  syphilis  and  a  similar  variation  is  noticeable  in  the  lower  animals. 
Young  monkeys  are  much  less  susceptible  than  adults  or  old  monkeys,  and 
among  some  species  while  the  adults  are  easily  infected  the  young  animals 
are  immune  (Roux  and  Metchnikoff). 

It  would  seem  that  the  virus  of  syphilis  can,  in  certain  cases,  become 
attenuated  by  passage  through  the  lower  monkeys  and  that  it  then  gives 
rise  in  the  higher  apes  and  perhaps  in  man  to  a  minimal  local  lesion  which 
is  not  followed  by  secondary  manifestations  and  which  moreover  immunizes 
the  inoculated  individual  against  a  second  inoculation.  Unfortunately,  the 
results  upon  which  this  conclusion  is  based  are  very  inconstant  as  will  be  seen 
from  the  accounts  of  a  few  typical  experiments  which  follow,  and  the  method 
cannot  be  regarded  as  a  sure  means  of  attenuating  the  virus. 

Metchnikoff  and  Roux  inoculated  a  chimpanzee  with  a  virus  from  a  bonnet 
monkey  (M.  sinicus}.  Small  insignificant  chancres  appeared  but  no  secondary 
lesions  and  the  chimpanzee  was  immune  to  a  subsequent  inoculation  with  a 
human  virus.  The  experiment  was  repeated  several  times  but  the  above 
result  was  never  again  obtained. 

Finger  and  Landsteiner  obtained  a  virus  which  after  passing  through  six 
baboons  (C.  hamadryas]  in  succession  set  up  in  a  seventh  an  insignificant 
lesion  which  lasted  only  a  short  time.  In  another  experiment  the  virus 
showed  no  attenuation  after  twelve  passages. 

Roux  and  MetchnikofE  passed  a  virus  from  a  chimpanzee  through  a  number 
of  M.  rhesus.  As  it  passed  from  rhesus  to  rhesus  the  primary  sore  became 
more  and  more  benign  and  appeared  earlier,  the  incubation  period  at  the 
same  time  falling  from  19—7  days.  After  eleven  such  passages,  the  virus  was 
very  attenuated  for  rhesus  monkeys  and  harmless  to  the  chimpanzee. 

Roux  and  Metchnikoif  inoculated  a  man  79  years  of  age,  and  who  was  not 
known  ever  to  have  had  syphilis,  on  the  fore-arm  with  an  human  virus  which 
had  been  passed  through  five  monkeys,  a  baboon,  two  chimpanzees,  and  two 
bonnet  monkeys  (M.  sinicus}.  Twelve  days  after  the  inoculation  two  insignifi- 
cant papules  appeared  which  lasted  only  a  few  weeks  and  were  unaccompanied 
by  any  other  lesion.  The  controls  (a  chimpanzee  and  a  bonnet  monkey) 
developed  typical  chancres  after  incubation  periods  of  23  and  31  days 
respectively. 

Sub-cutaneous  inoculation  of  anthropoid  apes  with  syphilitic  material  does 
not  produce  the  disease.  This  being  so  it  might  be  thought  that  sub-cutaneous 
inoculation  would  have  an  immunizing  effect  but  by  experiment  this  is 
found  not  to  be  the  case  ;  animals  inoculated  sub-cutaneously  are  just  as 
easily  infected  by  scarification  as  the  controls  (Metchnikoff,  Roux  and 
Neisser). 

The  syphilitic  virus  after  being  heated  to  51°  C.  has  no  immunizing  action 
when  inoculated  into  susceptible  animals. 

Serum  therapy. — Up  till  now  all  attempts  to  obtain  an  antisyphilitic  serum 
have  failed.  Roux  and  Metchnikoff  attempted  the  preparation  of  a  serum 
by  inoculating  macacus  monkeys  and  baboons  which  had  recovered  from 
a  primary  sore  with  large  quantities  of  a  virus  of  human  origin.  The  serum 
of  these  monkeys  sometimes  neutralizes  the  human  virus  in  vitro  :  a  mixture 


MORPHOLOGY  725 

of  serum  and  virus  is  without  effect  on  monkeys,  but  the  serum  alone  is 
useless  when  the  virus  has  been  already  inoculated.  No  immunizing  sub- 
stances are  present  in  the  blood  of  persons  suffering  from  the  disease  nor  in 
the  broken-down  tissues  of  gummata. 


SECTION  II.— MORPHOLOGY. 
1.  Microscopical  appearance. 

The  Treponema  pallidum  may  be  examined  in  the  fresh,  unstained  condition 
with  an  ordinary  microscope  if  a  powerful  source  of  light  (e.g.  an  inverted 
incandescent  burner)  (fig.  Ill,  p.  118)  and  a  good  oil-immersion  lens  be  used. 
Under  these  conditions,  the  organism  appears  as  a  small,  spiral  filament  with 
pointed  ends  and  exhibits  very  active  movements  of  rotation  and  flexion. 
The  spiral  arrangement  is  equally  evident  when  the  treponema  is  actively 
moving  and  when  it  is  at  rest :  the  spiral  is  complete  and  has  a  corkscrew 
appearance. 

The  examination  of  treponemata  in  the  fresh  condition  is  much  facilitated 


FIG.  344. — Treponema  pallidum.  Scraping  from  an  hard  chancre.  Dark- 
ground  illumination.  Epithelial  debris,  leucocytes,  bacteria  and  treponemata. 
(After  Gastou.) 

by  the  use  of  dark-ground  illumination  (p.  123)  which  is  also  of  great 
assistance  when  making  a  diagnosis.  With  the  ultra-microscope  the  trepo- 
nema appears  as  a  brilliant  spiral  standing  out  sharply  against  the  black 
background  of  the  field, of  the  microscope;  it  exhibits  more  or  less  rapid 
movements  of  progression,  and  seems  to  turn  rapidly  on  itself  like  a  screw 
and  to  move  like  an  eel.  Sometimes  the  treponemata  have  the  appearance 
of  "  a  series  of  brilliant  scintillating  points  travelling  one  behind  the  other 
in  a  straight  or  sinuous  line,  keeping  their  respective  distances  in  a  sort  of 
Indian  file  procession  "  (Gastou). 

When  stained,  the  treponema  measures  6-15/x  long  and  about  0'25/x  across. 
Occasionally  much  longer  forms  are  encountered ;  these  consist  of  several 
parasites  attached  to  each  other  end  to  end.  The  transverse  section  of  the 
organism  is  circular. 

The  turns  of  the  spiral  are  perfectly  regular  especially  about  the  centre  of 


726 


THE   SPIROCHJETE   OF   SYPHILIS 


the   parasite.     They  number  from  six  to  twelve,   though   occasionally  as 
many  as  twenty-six  have  been  seen.     At  each  end  there  is  a  filament  like  a 

bacterial  flagellum  which  may  be  about 
one-half  the  length  of  the  body :  these 
filaments  proceed  by  insensible  grada- 
tions from  the  body  of  the  organism  of 
which  they  seem  to  be  a  gradually 
vanishing  prolongation. 

An  undulating  membrane  is  never 
seen. 

Some  treponemata  appear  broader  than 
the  normal  with  a  bifurcated  end  and 
two  flagella  (fig.  345)  ;  many  of  these 
have  a  Y-shape  others  that  of  a  V. 

Occasionally  two  treponemata  are  seen 
attached  by  their  anterior  and  posterior 
ends,  but  separated  intermediately,  so 
as  to  give  the  appearance  of  an  elon- 
gated, irregularly-shaped  0.  Such  forms 
are  said  to  represent  stages  in  the 
longitudinal  division  of  the  organism 
(Schaudinn,  Prowazek,  and  others). 
Goldhorn,  Zettnow,  Levaditi  are,  however,  of  opinion  that  the  treponema 
divides  transversely  and  that  the  forms  just  described  are  due  to  two 
organisms  becoming  connected  together  by  their  ciliary  prolongations  or  at 
some  point  on  their  bodies. 


FIG.  345. — Treponema  pallidum.  a,  b, 
normal  forms;  c,  d,  e, —  0,  Y  and  F-shaped 
forms  ;  f,  three  treponemata  attached  end  to 
end. 


FIG.    346. — Treponema    pallidum.     Congenital    syphilitic    liver.     Giemsa's 
stain,      x  4000.     (From  the  Bulletin  de  I'Institut  Pasteur.) 


2.  Staining1  methods. 

The  Treponema  pallidum  stains  with  difficulty  and  never  other  than  very 
lightly,  hence  the  names  Spirochceta  pallida  and  Treponema  pallidum.  The 
organism  is  gram-negative. 

Special  methods  of  staining  have  to  be  adopted,  and  it  may  be  convenient 
to  record  those  which  are  likely  to  be  most  generally  met  with  in  works 
on  syphilis,  and  to  specify  those  of  them  which  appear  to  be  the  most 
useful. 


STAINING   OF  FILM  PREPARATIONS  727 

(«)  Staining  of  films. 

Giemsa's  methods.  A.  Slow  method.  Recommended.— 1.  Dry  the  films 
in  the  air  ;  fix  in  absolute  alcohol  for  half  an  hour. 

2.  Stain  for  20  hours  in  Petri  dishes  in  the  following  solution  : — 

Giemsa's  solution1  (Griibler),     '-  -          -      xxxv  drops.2 

Distilled  water,  20  c.c. 

3.  Wash    in    distilled   water.     Dry   with   filter   paper,    or   in   the   warm 
incubator.     Examine  with  an  oil-immersion  lens 

without  a  cover-glass,  or  mount  in  balsam. 
The  Spirochaetes  are  stained  reddish-violet. 

B.  Quick  method.    Recommended. — 1.  Fix  for 
30  minutes  in  absolute  alcohol ;  or  better,  for 
a   few   seconds  in  the   vapour   of    osmic  acid 
(Schaudinn). 

2.  Stain  for  1  hour  in  the  following  solution 
which  must  be  freshly  prepared  : — 

Giemsa's  solution, ...  10  drops 
1  per  cent,  aqueous  solution  of 

carbonate  of  sodium,  -          -  10      „ 

Distilled  water,      -         -         -  10  c.c. 

3.  Wash  in  distilled  water.     If  the  preparation        FiQ.Stf.—Treponcmapattidum. 
be  over-stained  leave  it  in  water  for  from  1-5      x1io(K)repar 

minutes.     Dry.     Examine. 

C.  Rapid  method  with  heat.     Recommended.     1.  Make  very  thin  films 
on  a  slide.     Dry.     Fix   by  passing  three   times  through   a   small    Bunsen 
flame. 

2.  Drop  a  large  drop  of  the  following  mixture,  recently  made  up  in  a  per- 
fectly clean  vessel,  on  to  the  slide. 

Distilled  water,    .....  10  c.c. 

Giemsa's  solution,         .....  10  drops. 

1  per  cent,  solution  sodium  carbonate,   -         -         -  5  to  10       „ 

Heat  the  slide  with  the  stain  on  it  for  a  few  seconds  over  a  small  flame 
until  steam  rises  but  avoid  boiling  the  solution. 

Pour  off  the  stain  and  flood  the  slide  with  a  fresh  quantity.  Heat  again. 
Change  the  stain  in  this  way  three  times  but  let  the  third  lot  of  stain  act  for 
1-2  minutes. 

3.  Wash  in  water.     Dry  with  filter  paper.     Mount  in  balsam.     Examine. 

The  methods  given  above  are  better  than  Giemsa's  original  method  which  was 
used  by  Schaudinn  in  his  early  work  on  the  spirochaste.  Schaudinn  fixed  his  prepara- 
tions as  above  and  then  stained  for  16-24  hours  in  the  following  solution  : 

Aqueous  solution  of  Giemsa's  eosin  (0*05  per  1000),  12  parts. 

Aqueous  solution  of  Azur  I.  (1  per  1000),         -  3       „ 

Aqueous  solution  of  Azur  II.  (0'8  per  1000),    -  2       „ 

Marino's  method.  Recommended. — This  is  a  rapid  method  and  does  not 
entail  fixing  the  films. 

1  Giemsa's  solution  may  be  obtained  ready  made  from  Griibler  of  Leipzic.     It  is  pre- 
pared by  mixing  : — 

Azur  II.  Eosin,    -          -  3        grams. 

Azur  II.,     -         -  0-80  gram. 

Glycerin,     -  -         250        c.c. 

60  per  cent,  methyl  alcohol,  -         350          ,, 

Dissolve  the  stains  in  the  alcohol  then  add  the  glycerin.  Leave  for  24  hours.     Filter. 

2  1  c.c.  — xxx  drops. 


728  THE   SPIROCILETE   OF   SYPHILIS 

1.  Pour  about  1  c.c.  of  the  following  solution  on  the  dried  preparation. 

Marino's  blue,1     -  O'lO  gram. 

Absolute  methyl  alcohol,       -  50        c.c. 

2.  Stain  for  about  3  minutes. 

3.  Without  washing,  pour  a  few  drops  of  a  O005  per  cent,  aqueous  solution 
of  eosin  on  the  film  and  leave  it  to  stain  for  1  or  2  minutes. 

4.  Wash.     Dry.     Mount  in  balsam. 

The  treponemata  will  be  stained  blue  and  violet. 

If  the  blue  has  precipitated  on  account  of  the  preparation  having  been  left  too 
long  in  the  staining  bath,  flood  the  film  with  the  blue  again  for  a  few  minutes,  then 
pass  through  eosin  for  1  minute  and  finally  wash. 

[The  above  solution  of  Marino's  blue  will  keep  for  about  2  months  if  the 
methyl  alcohol  be  pure.] 

Hecht  and  Wilenko's  method. — 1.  Place  a  drop  of  the  fluid  to  be  examined 
on  a  slide. 

2.  Add  a  drop  of  China  ink,  mix  carefully,  spread,  leave  to  dry  (half  an 
hour). 

3.  When  quite  dry,  examine  with  an  oil-immersion  lens.     The  treponemata 
appear  as  bright  structures  on  a  black  background. 

Ravaut's  method. — This  method  depends  upon  the  use  of  "  largine  " 
(albuminate  of  silver). 

1.  Make  the  films,  which  may  be  thick,  on  a  very  clean  slide.     Fix  in  methyl 
alcohol  or  in  osmic  acid  vapour. 

2.  Place  the  slide  in  a  stoppered  bottle  containing  the  following  solution, 
which  must  have  been  recently  made  up  and  kept  in  the  dark. 

Lillienf eld's  largine,      -          -  2  grams. 

Distilled  water,    -  100  c.c. 

Keep  the  slide  in  the  stain  at  55°  C.  for  2  hours. 

3.  Without  washing,  dip  the  slide  into  a  5  per  cent,  solution  of  pure  pyro- 
gallol.     The  silver  is  reduced  immediately. 

4.  Wash  in  distilled  water,  return  to  the  largine  bath  for  half  an  hour  and 
then  pass  through  the  pyrogallol  solution  again. 

5.  Wash.     Dry.     Examine.     The  treponemata  are  stained  deep  brown. 
The  foregoing  is  more  satisfactory  than  Stern's  silver  method.     Films  on  slides 

are  dried  in  the  air,  placed  in  the  37°  C.  incubator  for  a  few  hours,  then  transferred 
to  a  white  glass  bottle  containing  a  10  per  cent,  solution  of  silver  nitrate  and  exposed 
to  diffused  light  for  a  few  hours.  The  slides  which  now  have  a  metallic  appearance 
are  washed  in  running  water,  dried  and  examined.  The  treponemata  are  stained 
deep  brown  or  black. 

Borrel  and  Burnett's  method.  Recommended. — This  method  is  particularly 
useful  for  the  rapid  detection  of  the  treponema  and,  according  to  its  authors, 
is  more  certain  than  Giemsa's  method. 

1.  Excise  a  small  portion  of  the  infected  tissue  and,  with  the  point  of  a 
small  scalpel,  gently  scrape  a  little  of  it  on  to  a  number  of  slides  on  each  of 
which  a  small  drop  of  distilled  water  has  been  placed.     The  dissociated  tissue 
spreads  itself  out  in  the  water.     Dry. 

2.  Mordant  the  tissue  with  LcefEer's  ink  as  in  staining  for  flagella  (p.  150). 

1  [To  prepare  Marino's  blue  mix  : — 

Methylene  blue,  -  ....             o*5     gram 

Azur,  ...             0-5 

Water,  .         100        grams 
in  a  0-5  per  cent,  aqueous  solution  of  carbonate  of  soda.     Incubate  at  37°  C.  for  24-48 

hours.  Add  an  aqueous  solution  of  eosin.  Filter.  A  powder  is  thus  obtained  soluble 
in  water  and  methyl  alcohol.  ] 


STAINING   OF  FILM  PREPARATIONS  729 

3.  Stain  with  Ziehl's  fuchsin  as  for  flagella  (p.  151). 

Reitmann's  method. — This  is  an  application  of  Sclavo's  method  for  staining 
bacterial  flagella. 

1.  Fix  thin  films  in  absolute  alcohol  for  10  minutes. 

2.  Wash  in  distilled  water. 

3.  Wash  for  5  minutes  in  a  3  per  cent,  solution  of  phosphotungstic  acid. 

4.  Wash  in  water,  then  in  70  per  cent,  alcohol. 

5.  Stain  in  Ziehl's  fuchsin,  warming  until  steam  just  begins  to  rise. 

6.  Wash  in  70  per  cent,  alcohol,  then  in  distilled  water.     Dry. 
Herxheimer's  method. — 1.  Prepare  films  on  slides  and  fix  in  absolute  alcohol. 

2.  Stain  for  a  quarter-of-an-hour  in  an  aqueous  solution  of  gentian- violet  saturated 
in  the  warm. 

3.  Wash.     Dry.     Mount  in  balsam. 

Herxheimer  and  Huber's  method. — 1.  Fix  for  10  minutes  in  absolute  alcohol. 

2.  Stain  in  the  following  solution,  which  must  be  filtered  just  before  use,  for 
16-24  hours. 

Nile  blue  (or  Capri's  blue),  -  010  gram. 

Distilled  water,  -        100        c.c. 

3.  Wash  in  distilled  water.     Dry. 

Proca  and  Vasilescu's  method. — 1.  Fix  for  30  minutes  in  absolute  alcohol. 

2.  Immerse  in  the  following  mordanting  solution  for  10  minutes. 

Liquid  carbolic  acid,     -          -  50  grams. 

Tannin,       -  40       „ 

Distilled  water,    -  -         100 

Basic  fuchsin,       -  2 '5    „ 

Absolute  alcohol,  -         100      c.c. 

Mix  the  carbolic  acid,  tannin  and  water  and  then  add  the  fuchsin  after  dissolving  it  in 

the  alcohol. 

3.  Wash  in  distilled  water. 

4.  Stain  for  5  minutes  in  carbol-gentian- violet. 

5.  Wash  in  water.     Dry. 

Oppenheim  and  Sachs'  method. — No  preliminary  fixing  of  the  film  is  required  and 
the  treponemata  are  stained  without  having  been  dehydrated  in  alcohol,  with  the 
result  that  the  transverse  diameter  of  the  parasites  is  said  to  appear  larger  than 
when  they  are  treated  by  the  classical  methods. 

1.  Dry  the  film. 

2.  Without  fixing,  flood  it  with  the  following  solution  : 

Saturated  alcoholic  solution  of  gentian- violet,  10  c.c. 

5  per  cent,  carbolic  acid  in  water,  -         -         100     ,, 

Heat  the  stain  over  a  small  flame  until  steam  just  begins  to  rise. 

3.  Wash.     Dry.     Mount  in  balsam. 

Davidson's  method. — 1.  Dry  the  film.     Fix  in  absolute  alcohol  for  10  minutes. 

2.  Stain  for  1—10  hours  in  a  saturated  aqueous  solution  of  Muhleimer's  cresyl- 
violet  R  extra.     The  stain  must  be  freshly  prepared  and  filtered  before  use. 

3.  Wash  in  distilled  water.     Dry.     Mount  in  balsam. 

Zabolotny's  method. — Dry.  Fix.  Mordant  with  a  5  per  cent,  solution  of  carbolic 
acid.  Stain  in  the  warm  for  15  minutes  in  a  mixture  containing  01  per  cent,  azur 
solution  and  0*2  per  cent,  eosin  solution. 

Simonelli  and  Bandi's  method. — In  this  method,  as  in  Marino's,  preliminary 
fixation  is  eliminated,  the  stain  being  dissolved  in  methyl  alcohol. 

Method  of  preparing  the  stain. — Dissolve  1  gram  of  water-soluble  eosin  in  a  litre 
of  water,  and,  in  another  vessel,  1  gram  of  methylene  blue  in  a  litre  of  water.  Mix 
the  two  solutions  and  let  the  mixture  stand  for  a  week.  Filter  through  an  unfolded 
filter  paper  and  wash  the  precipitate  with  distilled  water.  Dry  the  precipitate  in 
the  air  then  dissolve  it  in  sufficient  methyl  alcohol  to  yield  a  saturated  solution. 

Method  of  staining. — 1.  Dry  the  film  and,  without  fixing,  stain  in  the  above 
solution  for  about  10  seconds. 

2.  Wash  very  quickly  in  distilled  water. 

3.  Blot  with  filter  paper.     Examine  without  a  cover-glass. 

Goldhorn's  method  is  merely  a  complicated  modification  of  the  preceding. 
Hoffmann  and  Halle's  method. — Fix  the  wet  film  with  osmic  acid. 


730  THE   SPIROCILETE   OF   SYPHILIS 

1.  Pour  into  a  small  capsule  of  about  5  cm.  diameter : 

1  per  cent,  aqueous  solution  of  osmic  acid,      -  5  c.c. 

Crystallized  acetic  acid,          ...     *    -  10  drops. 

Place  the  capsule  inside  a  glass  vessel  closed  with  a  ground-glass  cover. 

2.  Lay  a  well-cleaned  slide  on  the  capsule  for  a  couple  of  minutes. 

3.  Spread  the  material  to  be  examined  on  this  slide  with  a  platinum  spatula. 

4.  Then,  without  drying  the  film,  replace  the  slide  on  the  capsule  and  leave  it 
exposed  to  the  vapours  for  about  2  minutes. 

5.  Dry  the  film  quickly  by  gently  heating  it. 

6.  Flood  the  preparation  for  a  minute  with  a  weak  (pale  red)  solution  of  potassium 
permanganate. 

7.  Wash  in  distilled  water.     Blot  with  filter  paper. 

8.  Stain  with  Giemsa's  solution  as  described  above. 

9.  Wash.     Dry.     Examine. 

The  treponemata  stain  bluish-red  even  in  the  thick  parts  of  the  film.  The  flagella 
are  also  rendered  distinctly  visible. 

(P)  Flagellum  staining. 

The  flagella  of  the  Treponema  pallidum  may  be  stained  by  Loeffler's  original 
method  (p.  149)  or  modifications  of  it. 

Borrel  and  Burnett's  method  (p.  728)  gives  very  good  results  as  does  also 
Hoffmann  and  Halle's  method. 

Schaudinn  employed  Loeffler's  method,  thus  : 

1.  Fix  in  absolute  alcohol  for  10  minutes. 

2.  Mordant  with  fuchsin  ink  (p.  150)  heating  the  solution  gently  for  30-60 
seconds. 

3.  Wash  in  distilled  water,  then  in  75  per  cent,  alcohol. 

4.  Stain  with  gentle  heat  for  about  1  minute  in  a  large  drop  of  the  following 
solution  : 

Aniline  water,      ------  -         100  c.c. 

Basic  fuchsin,      -         -  -        Q.S.  to  saturate. 

1  per  cent,  soda  solution,       -  10  to  15  drops. 

5.  Wash  in  distilled  water.     Dry. 

(7)  Methods  of  staining  sections. 

Bertarelli,  Volpino,  and  Bovero's  method. — THese  observers  were  the  first 
to  successfully  stain  the  Treponema  pallidum  in  sections  of  syphilitic  tissues. 
The  method,  a  modification  of  van  Ermengem's  method  for  staining  flagella, 
is  based  upon  the  reduction  of  silver  nitrate  in  the  tissues  of  the  treponema. 
The  results  obtained  were,  at  the  time,  criticized  by  German  writers,  but 
there  can  now  be  no  doubt  as  to  the  true  nature  of  the  "  Spirochsetes  of  silver  " 
as  the  treponemata  were  described. 

1.  Fix  the  tissues  in  absolute  alcohol  (p.  188),  embed  in  paraffin  and  cut 
into  very  thin  (ca  5/u)  sections. 

2.  Immerse  the  sections  in  a  solution  of  silver  nitrate  for  24—48  hours. 

Crystals  of  silver  nitrate,       -         -         -         -         -         -  0*5  gram. 

Distilled  water,    -  -         100      c.c. 

3.  Wash  in  distilled  water. 

4.  Transfer  the  sections  to  van  Ermengem's  tanno-gallic  solution  (p.  149) 
for  a  quarter-of-an-hour. 

5.  Wash  in  distilled  water. 

6.  Return  the  sections  to  the  silver  solution  until  they  assume  a  brownish- 
yellow  colour. 

7.  Wash.     Dehydrate  in  absolute  alcohol.     Wash  in  xylol  and  mount  in 
balsam. 


STAINING   OF   SECTIONS  731 

The  spirochsetes  stain  black,  and  stand  out  prominently  against  the  deep 
yellow  colour  of  the  tissues. 

Levaditi's  methods. — In  order  to  avoid  the  deposits  which  are  often  formed 
when  Bertarelli's  method  is  used,  Levaditi  stained  the  tissue  in  bulk  in  the 
silver  solution.  Levaditi  has  described  two  methods,  the  second  of  which 
gives  the  better  results. 

First  method. — 1.  Fix  a  small  piece  of  tissue  in  10  per  cent,  formalin  for 
24  hours. 

2.  Harden  in  absolute  alcohol  for  24  hours. 

3.  Wash  in  distilled  water  for  5  minutes. 

4.  Immerse  the  tissue  in  a  solution  of  silver  nitrate  in  a  ground-glass 
stoppered  bottle  and  keep  it  in  the  dark  for  3  days  at  a  temperature  of  38°  C. 

Crystals  of  silver  nitrate,       -  1  '50  grams. 

Distilled  water,    ....  -         100        c.c. 

5.  Wash  in  water.     Then  immerse  for  24  hours  at  the  temperature  of  the 
laboratory  in  the  following  solution  : 

Pyrogallol,  4  grams. 

Formalin,  5  c.c. 

Distilled  water,    -  100    „ 

6.  Wash  in  distilled  water. 

7.  Dehydrate  in  80  per  cent,  alcohol,  then  in  absolute  alcohol,  clear  in  xylol 
and  embed  in  paraffin. 

8.  Cut  and  mount  the  sections  carefully  (p.  215)  and  stain  them  on  the 
slide  for  10  minutes  with  Giemsa's  solution  undiluted. 

9.  Wash  in  water  and  differentiate  in  absolute  alcohol  to  which  a  few  drops 
•  of  clove  oil  have  been  added. 

10.  Wash  in  absolute  alcohol,  oil  of  bergamot  and  xylol  and  mount  in 
balsam. 

The  re-staining  of  the  groundwork  with  Giemsa's  solution  (Stage  8)  is  not  essential  ; 
it  is  better  to  omit  it. 
Second  method  (Levaditi  and  Manouelian).    Recommended. — This  method 


< 
•1      , 


»  ~      w' 

^         r^ 

^s  J    ^-L 


FIG.  348. — Treponema  pallidum.     Section  of  lung.     Silver  impregnation  method. 
(Oc.  2,  obj.  TUh,  Zeiss.) 

differs  from  the  preceding  in  the  introduction  of  pyridine  which  shortens  the 
time  of  staining  and  facilitates  the  reduction  of  the  silver  salt. 


732  THE   SPIROCILETE   OF   SYPHILIS 

1.  and  2. — Cut  the  tissues  into  pieces  about  1-2  mm.  square,  then  fix  and 
harden  as  in  the  first  method. 

3.  Wash  in  distilled  water  until  the  pieces  fall  to  the  bottom  of  the  vessel. 

4.  Leave  the  pieces  of  tissue  in  a  ground-glass  stoppered  bottle  for  2-3 
hours  at  the  temperature  of  the  laboratory  then  for  3-5  hours  at  50°  C. 
in  50  c.c.  of  the  following  solution  : 

1  per  cent,  aqueous  solution  of  silver  nitrate,  -  90  e.c. 

Pyridine, 10    „ 

5.  Wash  in  distilled  water. 

6.  Immerse  for  3  or  4  hours  at  the  temperature  of  the  laboratory  in  the 
following  solution  which  must  be  freshly  prepared. 

4  per  cent,  aqueous  solution  of  pyrogallol,       -  90  c.c. 

Pure  acetone,       -         -  10     „ 

Pyridine,     -         -  17     „ 

7.  On  taking  out  of  the  reducing  bath,   dehydrate  in  absolute  alcohol, 
clear  in  xylol  and  embed  in  paraffin. 

8.  Cut,  stain  the  sections  on  the  slide  with  a  2  per  cent,  aqueous  solution 
of  toluidine  blue  (Buschke  and  Fischer)  and  differentiate  in  absolute  alcohol 
or  Unna's  ether-glycerin  solution.     (This  step  is  not  essential  but  by  adopting 
it  the  groundwork  is  stained  blue  and  the  relationship  of  the  parasites  to  the 
cells  can  be  made  out.) 

9.  Dehydrate  in  absolute  alcohol,  oil  of  bergamot  and  xylol.     Mount  in 
balsam.     The  treponemata  are  stained  black. 

SECTION  III.— THE  DETECTION  AND  IDENTIFICATION  OF  THE 
TREPONEMA  PALLIDUM  IN  THE  TISSUES. 

The  distribution  of  the  Treponema  pallidum  in  syphilitic  tissues  is  very 
irregular.  In  similar  lesions,  the  parasites  are  in  some  cases  numerous,  in 
others  so  few  in  number  that  several  preparations  have  to  be  examined  before 
one  or  two  are  seen.  Further,  a  delicate  technique  has  to  be  adopted,  so 
that  several  examinations  are  in  most  cases  essential. 

In  the  lesions  of  congenital  syphilis  the  treponemata  are  far  more  numerous 
than  in  the  lesions  of  acquired  syphilis.  In  the  latter  case  the  parasites  can 
most  easily  be  found  in  the  primary  lesions  and  in  the  secondary  papules. 
Treponemata  are  rarely  seen  either  in  the  blood  or  in  tertiary  lesions. 

Herscheimer  and  Opificus  advise  the  routine  use  of  preparations  made 
during  the  night  as  they  believe  that  the  micro-organism  is  then  present  in 
larger  numbers. 

1.  Collection  of  material. 

Chancre.— Cleanse  the  surface  of  the  chancre  carefully  to  expose  the  dermis. 
Scrape  it  gently  and  repeatedly  with  a  vaccinating  lancet  in  order  to  produce 
a  slight  exudation  of  serum,  a  sort  of  serous  dew,  and  prepare  films  with  the 
exudate  (Thibierge,  Ravaut  and  Le  Sourd). 

Alternatively,  the  chancre  may  be  partially  or  totally  excised  and  so  pro- 
vide material  for  cutting  sections.  This,  however,  may  not  always  be 
practicable. 

Rose  spots  and  papules.— The  simplest  method  is  to  scarify  the  part  and 
collect  the  exudate.  Better,  after  lightly  scarifying,  place  a  small  Bier's 
cupping  glass  (Zabolotny)  or  small  blister  over  the  lesion  and  use  the  serous 
fluid  which  collects. 

Gummata. — The  fluid  in  softened  gummata  contains  no  treponemata. 
Material  from  the  walls  of  gummata  must  be  used  for  the  purpose  of  demon- 
strating the  organism. 


DETECTION   OF   THE   PARASITE  733 

Lymphatic  glands. — Puncture  the  gland  with  a  large-bored  needle  and 
aspirate  the  juice  with  a  sterile  syringe. 

Blood. — Examination  of  blood-films  generally  gives  negative  results,  so 
that  it  is  desirable  to  select  one  or  other  of  the  following  special  methods  in 
searching  for  the  parasite. 

Nattan-Larrier  and  Bergeron's  method. — 1.  Collect  10  c.c.  of  blood  from  a 
vein  at  the  bend  of  the  elbow  (p.  193). 

2.  Distribute  the  blood  into  two  flasks  each  containing  100  c.c.  of  distilled 
water. 

3.  Let  the  blood  hsemolyze  and  then  centrifuge. 

4.  Prepare  thin  films  with  the  centrifuged  deposit.     Dry.     Fix  in  a  mixture 
of  alcohol-ether. 

5.  Stain   by   Bertarelli's   method   (p.   730)  ;     or   with   Heidenhain's   iron 
hsematoxylin. 

Ravaut  and  Ponselle's  method. — 1.  Let  the  blood  fall,  drop  by  drop,  into 
30  c.c.  of  distilled  water. 

2.  Collect  the  clot,  wash  it,  blot,  harden,  stain  by  Levaditi's  method  and 
cut  like  an  histological  preparation. 

Nseggerath  and  Staehelin's  method. — 1.  Collect  about  1  c.c.  of  blood  in  a 
tube  containing  a  0'33  per  cent,  aqueous  solution  of  acetic  acid. 

2.  Leave  the  blood  to  hsemolyze.     Centrifuge. 

3.  Prepare  films  with  the  deposit.     Dry.     Fix  and  stain  by  Giemsa's  slow 
method. 

2.  Methods  of  examination. 

In  examining  material  for  the  Treponema  pallidum  it  is  always  necessary 
to  bear  in  mind  that  in  many  syphilitic  affections  (ulcerated  chancres,  mucous 
plaques,  suppurating  gummata,  etc.)  other  micro-organisms  are  present  in 
addition  to  the  specific  parasite,  so  that,  if  a  spirochsete  be  found,  great  care 
must  be  exercised  to  make  certain  that  the  one  seen  is  in  fact  the  Treponema 
pallidum,  because  numerous  other  spirochsetes,  differing  more  or  less  from  the 
specific  spirochaete  of  syphilis,  are  often  found  in  the  tissues  (vide  infra). 

I.  Examination  of  fresh  material. — The  examination  of  fresh  material  is 
very  unreliable  ;    the  extreme  tenuity  of  the  organism  and  its  feeble  powers 
of  refraction  render  its  detection  difficult  and  demand  very  acute  powers  of 
observation  on  the  part  of  the  investigator.     It  was,  however,  by  this  method 
that  Schaudinn  discovered  the  treponema. 

Scholtz  recommends  examining  the  material  in  a  hanging-drop  preparation. 
The  sweated  serum  or  scrapings,  rubbed  up  in  a  drop  of  normal  saline  solution, 
are  examined  in  a  cell  or  between  a  slide  and  cover-glass.  Fluid  from  a 
pemphigus  bulla  and  blister  fluid  should  be  examined  in  the  same  way. 

The  material  must  be  examined  by  artificial  light  (an  inverted  incandescent 
burner  (p.  118)  is  very  useful)  and  with  an  oil-immersion  lens  (Levaditiand 
Roche). 

II.  Examination  with  dark-ground  illumination. — This  is  not  only  the  most 
rapid  but  perhaps  the  most  reliable  method  for  finding  the  treponemata 
(Landsteiner  and  Mucha,  Gastou). 

Arrange  the  dark-ground  illuminator  as  directed  on  p.  125.  If  an  oil- 
immersion  lens  be  used  place  a  diaphragm  in  the  objective,  but  a  high  power 
dry  lens  is  quite  suitable.  An  inverted  incandescent  burner  or  Nernst  lamp 
is  a  good  source  of  light.  Everything  being  in  order,  place  the  drop  of  fluid 
either  pure  or  diluted  in  a  little  normal  saline  solution  between  the  slide 
and  cover-glass  (for  details  vide  p.  127). 

Under  these  conditions,  the  treponema  stands  out  brightly  against  the 
black  back-ground  of  the  preparation  (fig.  344)  and  is  easily  seen.  The  most 


734 


THE   SPIROCILETE   OF   SYPHILIS 


difficult  part  of  the  experiment  is  to  differentiate  the  Treponema  pallidum 
from  the  various  similar  spirochsetes  which  are  not  infrequently  present  in 
these  preparations  (fig.  349).  A  certain  amount  of  experience  is  necessary 
before  it  is  possible  to  be  certain  of  the  diagnosis. 

III.  Examination  of  stained  preparations.  —  With  regard  to  the  staining  of 
films  and  sections  for  the  detection  of  the  treponema,  Borel  and  Burnett's 
method  is  rapid  and  is  particularly  recommended.  Giemsa's  method  is  also 
good.  For  sections,  the  silver  impregnation  method  is  undoubtedly  the 
best. 

3.  Identification  of  the  organism. 

The  Treponema  pallidum  is  differentiated  from  all  other  spirochsetes  by 
several  characteristics. 

1.  It  has  an  average  length  of  10-15/x  but  is  often  longer  and  is  extremely 
slender,  measuring  on  an  average  0'25/A  transversely. 

2.  Its  index  of  refraction  is  very  low 
indeed  in  fresh  preparations,  so  that  with 
an  ordinary  microscope  it  is  only  visible 
with  a  very  good  apochromatic  objective. 
On  the  other  hand,  it  is  readily  visible 
by  dark-ground  illumination. 

3.  It  has  no  undulatory  membrane. 

4.  Its  pointed  ends  terminate  in  a  long 
ciliary  filament. 

5.  It  is  circular  in  transverse  section. 

6.  It  is  in  form  a  complete  spiral  like 
a  corkscrew.     This   characteristic   spiral 
arrangement  is  seen  both  when  the   or- 
ganism is  in  motion  and  when  at  rest. 

7.  The  turns  of  the   spiral  are  deep, 

close  an,d  ™^-  The  "Banian  pomms 
a  considerable  degree  of  elasticity  so  that 
it  is  not  easy  to  deform  it. 

8.  In  vitro  its  vitality  is  low  so  that  when  watched  under  the  ultra-micro- 
scope the  movements  cease  in  5-6  hours  at  the  temperature  of  the  laboratory. 

9.  The  treponema  stains  with  difficulty  and  is  coloured  red  with  Giemsa's 
solution. 

Spirochsetes  closely  resembling  the  Treponema  pallidum. 

The  following  are  the  principal  spirochsetes  with  which  the  Treponema 
pallidum  is  likely  to  be  confused  : 

A.  Spirochaeta  refringens.—  This   spirochaete   occurs   in   smegma,   and   in 
ulcerating  lesions  of  the  skin.     It  is  sometimes  found  associated  with  the 
Treponema  pallidum  in  ulcerating  syphilitic  lesions,  but  in  these  cases  it 
occurs  near  the  surface  and  not  deep  in  the  tissues. 

The  Spirochceta  refringens  is  larger  and  longer  than  the  Treponema  pallidum 
and  in  the  fresh  condition  is  highly  refractile.  The  turns  of  the  spiral  are 
fewer  in  number  and  of  greater  amplitude,  less  regular  and  flattened.  The 
periplast  often  simulates  an  undulatory  membrane.  There  is  only  one 
flagellum  and  this  is  attached  laterally  to  one  of  the  rounded  ends  (Levaditi). 
The  movements  are  much  more  rapid  than  those  of  the  Treponema  pallidum 
and  it  is  often  impossible  to  follow  them  under  the  microscope.  It  stains 
easily  with  the  ordinary  dyes  and  stains  blue  with  Giemsa's  solution. 

B.  Spirochaeta     balanitidis.—  Hofmann    and     Prowazek     described     this 
organism  as  being  present  in  a  case  of  circinate  ulcerative  balanitis.     It 


Htic  papiiioma.     x  1500. 


DIFFERENTIATION  FROM   OTHER   SPIROCELETES       735 

would  appear  to  be  identical  with  the  Spirochceta  refringens,  from  which  it 
is  distinguished  only  by  minor  differences  in  the  arrangement  of  the  turns  of 
the  spirals.  It  occasionally  has  two  flagella,  attached,  as  in  the  case  of 
Spirochceta  refringens,  to  the  rounded  end  of  the  parasite.  This  is  probably 
the  organism  which  Levaditi  succeeded  in  growing  symbiotically  with  certain 
anaerobic  organisms  in  collodion  sacs  filled  with  heated  human  serum  in 
the  peritoneal  cavity  of  a  rabbit. 

C.  Spirochseta  plicatilis. — This  is  a  large,  thick  spirochsete  which  stains 
easily.     The  undulations  are  widely  separated  and  of  large  amplitude.     It 
has  a  large  undulatory  membrane  but  no  flagellum. 

D.  Spirochaeta   dentium. — This   spirochsete    which    multiplies    in    carious 
teeth  (Koch,  Miiller),  more  closely  resembles  the  Treponema  pallidum  than 
any  other  species  (Levaditi). 

In  common  with  the  Treponema  pallidum  it  is  an  organism  of  very  delicate 
structure  only  slightly  refractile  in  the  fresh  condition,  and  the  turns  of  the 
spiral  are  regular  and  permanent.  It  is,  however,  shorter  than  the  Treponema, 
its  average  length  being  4— 10/u,  and  the  turns  of  the  spiral  are  closer  together 
and  not  so  deep.  It  stains  more  easily  than  the  spirochsete  of  syphilis. 
Miihlens  and  Hartmann  have  been  able  to  grow  it  outside  the  body. 


FIG.  350. — Various  spirochaetes.     A,  Treponema  pallidum  :  B,  »S.  refringens  :  C,  Spirochsete 
from  a  cancer  :   D,  S.  plicatilis  :   E,  S.  dentium  :   F.  S.  vincenti. 

Miihlens  grew  it  in  Veillon's  tubes  containing  a  mixture  of  two  parts  of  liquefied 
agar  and  one  part  of  horse  serum  heated  to  58-60°  C.  for  half  an  hour.  The  serum- 
agar  was  sown  at  40°  C.  and  the  tubes  rapidly  cooled.  After  incubating  at  37°  C. 
for  8  days  very  small,  whitish,  transparent,  snow-like  colonies  were  seen  in  the 
depth  of  the  agar.  It  was  possible  to 'sow  a  series  of  sub-cultures.  The  cultures 
were  not  pathogenic.  The  spirochsetes  occasionally  had  a  long,  fine,  terminal 
flagellum  but  often  no  flagellum  at  all  could  be  seen.  No  undulatory  membrane 
was  found.  In  two  of  the  cultures  longitudinal  multiplication  forms  having  the 
form  of  a  Y  or  a  V  were  observed. 

E.  Spirochseta  buccalis. — The  Spirochceta  buccalis  was  described  by  Cohn 
as  occurring  in  the  human  mouth  ;  it  is  a  large  spirochsete  ;   the  undulations 
are  few  in  number  and  of  wide  amplitude  :    it  stains  easily,  and  has  one  or 
two  flagella  arranged  like  those  of  the  Spirochceta  refringens.     The  undulatory 
membrane  which  has  sometimes  been  described  in  connexion  with  it  would 
appear  to  be  formed  by  the  debris  of  the  periplast.     There  should  be  little  or 
no  difficulty  in  distinguishing  between  this  spirochsete  and  the  Treponema 
pallidum. 

F.  Spirochaeta  vincenti. — This  spirochsete,  found  in  association  with  fusiform 
spirilla  (p.  574),  has  the  same  characters  as  the  Spirochceta  buccalis.     It  must 
be  regarded  as  either  very  closely  related  to  or  identical  with  that  organism 
(Spirochceta  media  Prowazek). 

G.  The  Spirochsetse  of  malignant  ulcers. — In  malignant  ulcers,  Lcewenthal 


736  THE   SPIROCELETE   OF   SYPHILIS 

found  two  species  of  spirochaetes  quite  different  from  the  Treponema  pallidum. 
They  both  stain  blue  by  Giemsa's  method  and  in  both  the  undulations  are 
irregular  and  flat. 

Spirochceta  microgimta  is  small  and  delicate  measuring  2'5-6/^.  It  has 
four  to  twelve  turns  in  the  spiral  and  these  are  so  close  together  as  to  appear 
to  touch.  It  stains  well  with  borax-blue. 

Spirochceta  Icewenthali  is  thicker  than  the  Treponema  ;  the  turns  of  the 
spiral  are  irregular  and  it  seems  to  have  an  undulatory  membrane. 

H.  Treponema  pallidulum  (Spirochceta  pertenuis}. — Yaws  or  Frambcesia 
(Fr.  Pian)  is  a  contagious  and  inoculable  disease  very  common  in  the  tropics 
and  characterized  by  papillomatous  lesions  which  do  not  affect  the  mucous 
membranes.  It  is  caused  by  an  organism  very  closely  related  to  that  of 


FIG.  351 . — Scraping  from  an  infected  mucous  papule.     Dark-ground  illumina- 
tion.    (After  Gastou.)     Various  spirochsetes. 

syphilis,  and  was  first  described  by  Castellani  under  the  name  Spirochceta 
pallidula. 

The  following  characteristics,  according  to  Castellani  and  Prowazek,  serve 
to  identify  the  Treponema  pallidulum  :  it  is  a  little  thicker  than  the  Treponema 
pallidum  :  the  turns  of  the  spiral  are  rather  crowded  and  irregular  :  its  ends, 
often  coiled  up  together,  are  usually  rounded  and  a  terminal  flagellum  is  not 
a  constant  feature. 


SECTION  IV.— CULTIVATION  EXPERIMENTS. 

[Until  recently]  it  has  not  been  possible  to  grow  the  Treponema  pallidum 
in  artificial  media. 

Volpino  and  Fontana  cut  off  under  aseptic  precautions  fragments  of 
syphilitic  lesions  (chancres,  papular  lesions)  and  sowed  the  pieces  both 
aerobically  and  anaerobically  either  in  human  blood,  citrated  human  blood, 
serum  or  ascitic  fluid  and  incubated  the  tubes  at  37°  C.  Various  contaminat- 
ing organisms  grew  in  the  culture  fluid  but  in  no  case  was  a  culture  of  the 
treponema  obtained  ;  in  the  fragments  of  the  tissues  themselves  on  the 
other  hand  a  considerable  multiplication  of  spirochaetes  was  demonstrated 
after  about  8-10  days.  Even  when  no  spirochsetes  could  be  found  in  the 


CULTIVATION   OF  THE  SPIROCILETE  737 

tissue  before  sowing  they  were  readily  found  in  it  subsequently  and  had 
evidently  multiplied  in  the  culture  tubes. 

Levaditi  and  Macintosh  have  grown  the  treponema  in  collodion  sacs. 
The  virus,  obtained  from  monkeys,  was  sown  in  sacs  filled  with  human  blood, 
and  in  these  sacs,  placed  in  the  peritoneal  cavity  of  a  Macacus  cynomolgus, 
an  impure  culture  of  the  treponema  was  obtained.  Sub-cultures  were 
grown  in  series  by  placing  the  sacs  in  the  peritoneal  cavities  of  rabbits  ;  they 
contained  numerous  anaerobic  organisms  were  devoid  of  virulence  and  had 
no  immunizing  properties.  All  attempts  to  isolate  the  treponema  in  pure 
culture  failed. 

[Noguchi1  has  devised  a  method  by  which  he  has  been  able  to  grow  the 
Treponema  pallidum  in  pure  culture  outside  the  body.  Moreover  the  inocula- 
tion of  these  pure  cultures  has  resulted  in  the  development  of  typical  syphilitic 
lesions  in  the  rabbit. 

[Medium. — After  trial  of  many  culture  media  Noguchi  finds  that  the  most  suitable 
consists  of  a  mixture  of  1  part  of  sheep,  horse  or  rabbit  serum  with  3  parts  of  distilled 
water  into  which  a  piece  of  freshly  excised  normal  rabbit  tissue  (kidney  or  testicle) 
is  placed. 

[The  medium  is  tubed  in  quantities  of  16  c.c.  in  each  tube  and  sterilized  for  15 
minutes  on  each  of  3  successive  days  at  100°  C.  A  layer  of  sterile  paraffin  oil  is 
poured  on  the  surface  to  render  the  medium  anaerobic  and  to  prevent  evaporation. 
The  sterility  of  the  contents  of  the  tubes  is  determined  by  incubating  the  latter  at 
37°  C.  for  2  days. 

[Material. — For  sowing  the  medium  Noguchi  used  portions  of  the  testicles  of 
rabbits  which  had  been  infected  with  material  from  cases  of  syphilis  in  the  human 
subject.  By  using  rabbit  tissues  the  difficulty  of  contaminating  organisms  was 
largely  overcome. 

[Conditions  of  growth. — For  primary  cultivations  it  is  essential  that  the  medium 
shall  be  incubated  under  anaerobic  conditions.  The  tubes  may  be  placed  in  a 
Bulloch's  apparatus  which  is  then  exhausted  over  pyrogallol  and  the  bell  jar  after- 
wards filled  with  hydrogen. 

[The  serum  and  tissue  forming  the  culture  medium  must  be  slightly  alkaline. 

[To  remove  contaminations  Noguchi  filters  through  Berkefeld  filters  as  he  finds 
that  the  treponemata  are  filtrable  after  the  fifth  day. 

[Results. — The  rabbits  providing  the  material  for  cultivation  had  been  inoculated 
with  material  from  ten  different  sources.  Cultivations  were  obtained  from  six  of 
these.  Cultivation  was  difficult  and  many  failures  were  experienced.  Once  only 
did  Noguchi  obtain  a  pure  culture  at  first  trial.  One  strain  had  passed  through 
twenty-five  sub-cultures. 

[The  treponemata  begin  to  multiply  after  about  48  hours'  incubation  and  con- 
tinue slowly  to  increase  for  4  or  5  weeks.  They  attain  their  natural  size  in  10-12 
days  and  later  elongate  and  form  tangled  masses. 

[In  appearance  the  cultivated  treponemata  are  quite  typical  and  quite  indis- 
tinguishable from  treponemata  obtained  from  human  or  experimental  animal 
sources. 

[With  two  of  the  cultivated  strains  Noguchi  was  able  to  produce  lesions  in  the 
testicles  of  rabbits  which  in  every  way  resembled  the  lesions  produced  by  the  inocula- 
tion of  material  from  cases  of  human  syphilis.  ] 

SECTION  V.— SERUM  DIAGNOSIS. 

Wassermann,  Neisser  and  Bruck  conceived  the  idea  of  applying  to  the 

diagnosis  of  syphilis  the  complement  fixation  method  of  Bordet  and  Gengou. 

As  it  was  impossible  to  obtain  cultures  of  the  Treponema  pallidum,  Wassermann 

used  as  the  antigen  the  liver  of  a  congenitally  syphilitic  infant  which,  as  is 

well  known,  contains  very  large  numbers  of  the  organism.     He  showed  that 

an  extract  or  fragment  of  such  a  liver  will,  in  the  presence  of  heated  syphilitic 

f1  Journal  of  Experimental  Medicine,  vol.  xiv.] 

3A 


738  THE   SPIROCILETE   OF   SYPHILIS 

serum  and  guinea-pig  complement,  fix  the  complement ;  hence  the  conclusion 
that  in  syphilis  the  serum  contains  anti-bodies.  Wassermann  devised  a 
clinical  method  for  the  diagnosis  of  syphilis  based  on  this  observation  and 
deduction.  It  may  be  stated  as  follows  : — 

1.  If  an  hsemolytic  system  (p.  232)  be  added  to  a  mixture  of  extract  of 
syphilitic  liver,  heated  syphilitic  serum  and  complement,  no  haemolysis  occurs. 

2.  If  an  hsemolytic  system  be  added  to  a  mixture  consisting  of  the  same 
extract  of  liver,  heated  non-syphilitic  serum  and  complement,  hsemolysis 
occurs — there  has  been  no  fixation  of  complement. 

3.  Further,  if  the  cerebro-spinal  fluid  from  a  case  of  general  paralysis  or 
tabes  (diseases  which  are  known  to  be  of  syphilitic  origin)  be  mixed  with  an 
extract  of  a  syphilitic  liver  and  complement  the  latter  is  fixed  as  in  the  case 
of  a  syphilitic  serum. 

Experience  has  shown  that  there  are  certain  exceptions  to  the  above  rules. 
The  reaction  is,  as  a  rule,  positive  in  the  secondary  stage  of  syphilis  where 
lesions  are  present  (65-80  per  cent.).  The  percentage  of  cases  in  which  positive 
results  are  obtained  is  smaller  in  primary  syphilis,  and  also  and  especially 
when  the  disease  is  of  long  standing  and  when  at  the  time  of  examination  no 
symptoms  are  manifest  (20-58  per  cent.).  It  is  often  negative  in  cases — 
even  when  they  are  of  recent  origin — which  are  being  treated  with  mercury 
(ca  30  per  cent.).  Finally,  experiments  have  been  recorded  in  which  a 
positive  reaction  has  been  obtained  with  the  serum  of  persons  free  from 
syphilis  and  with  the  serum  of  non-infected  monkeys. 

The  reaction  is  therefore  only  reliable  when  it  is  distinctly  positive.  A 
negative  reaction  merely  constitutes  a  presumption  in  favour  of  the  non- 
existence  of  syphilis. 

In  general  paralysis,  Wassermann's  reaction  is  nearly  always  positive  if 
the  cerebro-spinal  fluid  be  used  in  the  test  (93  per  cent.),  but  often  negative 
if  the  blood  serum  be  employed  (58  per  cent.).  It  follows  therefore  that  the 
cerebro-spinal  fluid  should  always  be  used  in  these  cases. 

The  nature  of  Wassermann's  reaction. — Wassermann's  reaction  is  not,  how- 
ever, to  be  explained  on  the  assumption  that  syphilitic  anti-bodies  are  present : 
Armand-Delille,  Levaditi  and  Marie,  and  others  repeating  Wassermann's 
experiments  were  soon  able  to  show  that  the  same  results  are  obtained  when 
an  extract  of  the  liver  of  a  new-born  but  non-syphilitic  infant  is  substituted 
for  the  liver  of  a  syphilitic  infant.  This  observation  proves  that  there  is  no 
analogy  between  the  extract  of  syphilitic  liver  and  a  true  antigen.  And 
Levaditi  and  Marie  have  also  shown  that  the  cerebro-spinal  fluid  in  general 
paralysis  which  gives  the  Wassermann  reaction  contains  no  true  syphilitic 
anti-bodies  ;  for  it  is  incapable  of  destroying  or  diminishing  the  virulence 
of  the  treponema  in  vitro. 

Finally  Landsteiner,  Levaditi  and  Yamanouchi,  Forges  and  others  have 
shown  that  the  reason  why  the  liver  extract,  in  the  serum-reaction  in  syphilis, 
is  able  to  fix  the  complement  is  because  it  contains  certain  well-defined  sub- 
stances soluble  in  alcohol — lecithin  on  the  one  hand,  and  bile  salts  on  the 
other.  In  Wassermann's  reaction  the  extract  of  liver  can  be  replaced  by  an 
emulsion  or  solution  of  these  substances. 

Levaditi  and  Koche  conclude  that  the  serum-reaction  in  syphilis  would  not 
appear  to  be  due  to  the  interaction  of  antigen  and  anti-body  in  the  ordinarily 
accepted  meaning  of  those  terms  and  is,  further,  in  no  way  connected  with 
the  presence  of  the  treponema.  The  fact  would  appear  to  be  rather  that 
during  an  attack  of  syphilis  "  the  serum  becomes  enriched  in  certain  colloidal 
principles  which  in  presence  of  lipoids  and  bile  salts  are  easily  precipitated 


WASSERMANN'S  REACTION  739 

and  so  fix  the  hsemolytic  complement."  There  is  simply  an  increase  in  the 
body  fluids  of  substances  already  present  though  in  smaller  quantity  in 
normal  serum  ;  proof  of  this  is  afforded  by  the  fact  that  normal  serum  used 
in  large  doses  will  give  Wassermann's  reaction. 

The  serum-reaction  in  syphilis  is,  however,  notwithstanding  the  theories 
advanced  in  explanation  of  the  phenomenon,  frequently  used  as  a  practical 
method  for  the  diagnosis  of  syphilis.  The  experiments  summarized  above 
have  much  simplified  the  technique  of  the  reaction  and  a  purely  chemical 
test  depending  solely  upon  the  precipitation  or  non-precipitation  of  the  serum 
in  presence  of  certain  substances  and  especially  of  bile  salts  has  been  devised. 

Space  will  not  allow  a  description  of  all  the  modifications  of  Wassermann's 
method  which  have  been  introduced  ;  his  original  method  therefore,  and  the 
methods  based  upon  a  chemical  reaction  which,  by  reason  of  their  simplicity, 
are  now  at  the  disposal  of  all  medical  men,  will  alone  be  described. 

Wassermann's  technique  for  the  serum-diagnosis  of  syphilis. 

The  ordinary  reagents  used  in  the  Bordet-Gengou  reaction  are  necessary 
for  the  serum-diagnosis  of  syphilis. 

I.  Antigen. — -Wassermann  used  an  extract  of  the  liver  of  a  newly-born 
congenitally  syphilitic  infant.  An  extract  of  the  liver  of  a  newly-born  non- 
syphilitic  infant  serves  the  purpose  equally  well. 

(a)  A  portion  of  the  liver,  which  must  be  fresh  and  have  been  collected 
with  all  precautions  to  avoid  contamination,  is  ground  up  very  finely  with  a 
very  small  quantity  of  sterile  normal  saline  solution  in  a  Borrel's  mincer  or 
in  an  agate  mortar.  The  emulsion  is  poured  into  Petri  dishes  and  dried  in 
vacuo  over  sulphuric  acid.  The  dried  contents  of  the  Petri  dishes  are  col- 
lected and  ground  up  in  a  pepper  mill.  The  brown  powder  is  sifted  and  stored 
in  the  dark  in  absolutely  dry,  well-stoppered  bottles. 

When  required  for  use,  take  1  gram  of  the  powder  and  triturate  it  in  an 
agate  mortar  with  25  c.c.  of  normal  saline  solution.  Leave  the  emulsion  to 
stand  for  10  hours  in  the  ice  chest,  centrifuge  and  use  the  supernatant  liquid 
as  the  antigen. 

(6)  An  alcoholic  extract  of  the  liver  may  be  used  (Landsteiner).  One  part 
of  the  dried  and  powdered  liver  is  placed  in  a  stoppered  bottle  with  30  parts 
of  absolute  alcohol.  Leave  for  2  days  shaking  frequently  and  then  filter 
through  paper.  For  use,  dilute  this  extract  with  ten  times  its  volume  of 
water. 

In  the  same  way,  an  extract  of  fresh  liver  can  be  made  by  macerating  10 
grams  of  ground  up  liver  in  100  grams  of  absolute  alcohol. 

(c)  Levaditi  and  Yamanouchi,  instead  of  extract  of  liver,  use  the  following 
solution  : 

Taurocholate  or  glycocholate  of  sodium,  1        gram. 

Normal  saline  solution,  -         100        grams. 

Carbolic  acid,       -  0'50  gram. 

However  prepared,  the  antigen  must  be  carefully  titrated  before  being 
used.  For  this  purpose  the  experiment  will  be  arranged  as  described  on  p. 
235,  using  quantities  of  0*05  c.c.,  O'l  c.c.,  0'2  c.c.,  0'3  c.c.,  0'4  c.c.,  and  0!5  c.c., 
of  liver  extract.  In  the  subsequent  experiments  the  smallest  quantity  of 
antigen  which  will  give  complete  haemolysis  is  used  (0*2  c.c.,  for  example). 

II.  Suspected  serum  (anti-body). — -The  patient's  serum  collected  with  the 
aid  of  a  Bier's  cupping  glass  or  by  puncturing  a  vein  at  the  bend  of  the  elbow, 
is  left  to  coagulate.  The  serum  is  then  separated  and  heated  (inactivated) 
for  half  an  hour  at  55°-56°  C.  It  also  must  be  titrated  in  the  usual  manner 
(p.  236) 


740 


THE   SPIROCILETE   OF   SYPHILIS 


In  cases  of  general  paralysis  cerebro-spinal  fluid  must  be  used  in  place  of 
blood  serum.  This  fluid  does  not  need  to  be  heated  because  it  contains  no 
complement. 

III.  Complement. — Use  guinea-pig  complement  prepared  and  titrated  as 
described  at  p.  235. 

IV.  Haemolytic  system.— Use  sheep  red  cells  and  heated  (inactivated)  anti- 
sheep  rabbit  serum  (p.  234). 

Experimental  data. 

The  reagents  being  prepared  and  titrated  it  is  necessary,  in  order  that  the 
test  may  be  quite  reliable,  to  have  in  addition  some  serum  from  a  person 
suffering  from  syphilis  which  is  known  to  give  a  distinctly  positive  reaction 
and  some  serum  from  a  non-syphilitic  person  which  gives  a  negative  reaction. 
In  this  way  a  series  of  controls  is  available  for  comparison  and  the  chances 
of  error  are,  as  far  as  possible,  avoided. 

The  experiment  will  be  arranged  as  follows  in  accordance  with  the  rules 
laid  down  on  p.  236. 


Tube 
No.  1 
Tube 
No.  2 

I.  —  Mix  and  incubate  for  one  hour  and  a  half 
at  37°  C. 

II.  —  Add  after  an  hour 
and  a  half  and  incu- 
bate half  an  hour  at 
37°  C. 

RESULTS. 

Extract  of 
liver. 

Heated 

suspected 
serum. 

°S3f   \    ™ 

ment-         solution. 

Emulsion 
of 
red  cells. 

Heated 
hsemolytic 
serum. 

If  the  serum 
is 
syphilitic. 

If  the  serum 
is  not 
syphilitic. 

0-20  c.c. 
0'30  c.c. 

0'20  C.C. 

0-20  c.c. 

O'lO  c.c. 
0-10  c.c. 

0'40  c.c. 
0-30  c.c. 

1  C.C. 

1  c.c. 

O'lO  c.c. 
0-10  c.c. 

No 
haemolysis. 
No 
haemolysis. 

Complete 
haemolysis. 

Haemolysis. 

Control 
Control 

0-20  c.c. 
0-30  c.c. 

Nil. 
Nil. 

0-10  c.c. 
(HOc.c. 

0'60  c.c. 
0-50  c.c. 

1  c.c. 
1  c.c. 

0-10  c.c. 
0-10  c.c. 

Complete 
haemolysis 

Control 

Nil. 

0-20  c.c. 

0-10  c.c. 

0-60  c.c. 

1  c.c. 

0-10  c.c. 

Complete 
haemolysis. 

Control 

Nil. 

Nil, 

0-10  c.c. 

0-80  c.c. 

1  c.c. 

0-10  c.c. 

Complete 
haemolysis. 

Chemical  methods  of  serum  diagnosis. 

These  methods  are  based  on  the  fact  that  certain  reagents  while  having  no 
action  on  normal  serum  produce  a  precipitate  when  brought  in  contact  with 
syphilitic  serums.  The  results  are  perhaps  less  constant  than  those  obtained 
by  Wassermann's  reaction. 

Methods  of  Forges  and  Meyer. — Syphilitic  serum  generally  produces  a  pre- 
cipitate when  mixed  with  lecithin  whereas  normal  serum  does  not  under 
similar  circumstances  produce  a  precipitate. 

Technique.— 1.  Triturate  in  an.  agate  mortar  0*20  gram  of  ovo-lecithin 
(Merck)  adding  in  small  quantities  at  a  time  100  c.c.  of  normal  saline  solution. 


CHEMICAL   METHODS   OF  DIAGNOSIS  741 

2.  Take  a  number  of  test-tubes  (6-7  mm.  diameter)  and  pour  into  each 
1  c.c.  of  the  lecithin  emulsion  and  1  c.c.  of  the  suspected  serum.     As  a  control 
prepare  similarly  a  number  of  tubes  but  using  normal  serum.     Incubate  the 
tubes  for  from  i-6  hours  at  38°  C. 

3.  On  taking  the  tubes  out  of  the  incubator  leave  them  to  stand  at  the 
temperature  of  the  laboratory  before  recording  the  results.     The  Lubes  to 
which  the  syphilitic  serum  has  been  added  will  show  a  precipitate  which 
appears  first  at  the  surface. 

Forges'  method.    Method  recommended. — The  reaction  in  this  case  depends 
upon  the  use  of  a  solution  of  glycocholate  of  sodium. 

1.  Prepare  immediately  before  use  a  solution  consisting  of : 

Sodium  glycocholate  (Merck),         -  1  gram. 

Distilled  water,    -  -         100  c.c. 

2.  Heat  the  suspected  serum  for  half  an  hour  at  55°-56°  C. 

3.  To  each  of  a  series  of  small  test-tubes  add  : 

Heated  serum,     -  1  c.c. 

Glycocholate  solution,  1     ,, 

Prepare  similarly  a  number  of  tubes  with  heated  normal  serum. 

4.  Leave  the  tubes  at  the  temperature  of  the  laboratory  for  20  hours. 
A  precipitate,  most  distinct  at  the  surface  of  the  mixture,  is  formed  only  in 
those  tubes  containing  the  syphilitic  serum. 

Klausner's  method. — Distilled  water  produces  a  precipitate  when  mixed 
with  syphilitic  serum. 

1.  To  a  number  of  small  test-tubes  add  : 

Suspected  serum,  0*2  c.c. 

Distilled  water,    -  0'7     „ 

Prepare  a  number  of  tubes  with  normal  serum. 

2.  Leave  the  tubes  for  a  few  hours  at  the  temperature  of  the  laboratory 
and  then  examine  the  reaction.     The  syphilitic  serum  alone  produces   a 
distinct  precipitate  and  renders  the  mixture  cloudy. 


PART   V. 
THE   PROTOZOAN  PARASITES. 


CHAPTER  LV. 
THE   AMOEBA. 

Introduction. 

Section  I. — Amoeba  princeps,  p.  745. 

Section  II. — The  intestinal  amoebae,  p.  746. 

Introduction,  p.  746. 

Microscopical  appearance,  p.  747. 

I.  Amoeba  coli,  p.  747.     II.  Amoeba  histolytica,  p.  748. 

Methods  of  detection.     Staining  methods,  p.  748. 

Cultivation,  p.  750. 

Experimental  infection,  p.  751. 

FOB  some  years  past  the  Protozoa  have  assumed  a  position  of  considerable 
importance  in  human  and  veterinary  pathology.  In  this  and  the  following 
chapters  the  various  pathogenic  species  will  be  briefly  described  and  the 
methods  suitable  for  their  detection  and  study  indicated,  but  all  reference 
to  the  classification  and  biology  of  the  Protozoa  will  be  omitted.  For  these 
the  reader  is  referred  to  treatises  devoted  to  the  study  of  Protozoology. 

Among  the  Rhizopoda,  the  Amoeba)  alone  are  of  interest  from  the  point  of 
view  of  pathology.  Amoebae  are  frequently  found  in  the  human  intestine, 
and  one  species  is  the  cause  of  the  endemic  dysentery  of  warm  climates  ; 
other  species  have  been  found  in  ulcerations  of  the  mouth,  in  dental  tartar 
(Amoeba  buccalis  ;  Gross,  Sternberg,  Kartulis),  in  haematuria,  cystitis  and 
metritis  (Amoeba  urogenitalis  vel  vaginalis  ;  Boeltz,  Rossi  Doria  and  others) 
and  in  the  fluid  of  some  malignant  abdominal  tumours  (Miura,  Lieberkiihn, 
Leyden). 

Before  embarking  upon  a  study  of  the  pathogenic  amoebae  it  will  be  as 
well  to  acquire  a  certain  amount  of  practice  in  observing  these  protozoa,  and 
for  this  purpose  the  Amoeba  princeps,  a  very  widely  distributed  saprophytic 
species,  may  be  used. 


SECTION  L— AMCEBA   PRINCEPS. 

The  Amoeba  princeps  is  not  only  a  very  suitable  species  for  purposes  of 
observation,  but  specimens  can  be  readily  obtained  by  macerating  a  little 
straw  in  a  vessel  of  water.  In  such  an  infusion  numerous  bacteria  will  be 
found,  and  in  addition  to  other  Protozoa,  amoebae  appearing  as  large  masses 
(100/x  in  diameter)  of  granular  protoplasm  can  be  seen  after  a  few  days. 

The  amoeba  consists  of  an  hyaline  ectosarc  surrounding  a  granular  endosarc  which 
contains  several  contractile  vacuoles,  and  a  rounded  refractile  nucleus  which  can  be 


746 


THE   AMOEBAE 


rendered  more  conspicuous  by  treating  with  acetic  acid.     The  nucleus  stains  deeply 
with  ammoniacal  picro-carmine  while  the  protoplasm  is  only  lightly  tinted. 

The  amoeba  is  an  highly  mobile  organism  which  alters  its  shape  by  the  protrusion 
and  retraction  of  pseudopodia ;  by  successively  altering  its  shape  it  is  able  to  move 
from  place  to  place  and  to  collect  food  material.  A  solid  particle  with  which  one 
of  the  pseudopodia  may  have  come  in  contact  is  gradually  surrounded  and  enfolded 
by  the  organism  and  passes  into  the  interior  of  the  protoplasm  ;  if  it  be  suitable 


FIG.  352. — Amoeba  princeps.  Different  shapes  assumed  by  the  protozpon 
in  moving  across  the  field  of  the  microscope.  (Duration  of  observation, 
35  minutes.) 

for  food  it  is  gradually  dissolved  in  the  substance  of  the  protoplasm,  and  if  not 
suitable  it  is  soon  thrown  out.  Intra- cellular  digestion  is  accompanied  by  the 
secretion  of  acid  in  the  interior  of  the  protoplasm  (Metchnikoflf). 

Fig.  352  shows  the  different  shapes  assumed  by  an  Amoeba  princeps  in  the  field 
of  the  microscope  while  under  observation  for  35  minutes. 

Reproduction  takes  place  in  two  ways  : — When  conditions  are  favourable  the 
amoeba  divides  into  two,  the  nucleus  dividing  first  and  the  protoplasm  afterwards 
(schizogony}. 

But  when  the  medium  in  which  the  amoeba  is  living  begins  to  dry  up,  the  protozoon 
becomes  encysted  and  enters  upon  a  latent  existence.  During  the  encysted  stage 
the  nucleus  may  divide  into  several  secondary  nuclei  around  which  the  protoplasm 
collects,  forming  spores  (sporogony}.  When  the  conditions  again  become  favourable 
the  protecting  envelope  is  lost  and  the  animal  assumes  its  former  characteristics. 

The  Amoeba  princeps  can  be  readily  cultivated  in  infusions  of  hay  or  straw 
(p.  37),  on  similar  infusions  solidified  with  agar  or  on  a  jelly  of  Fuscus  crispus 
(5  per  cent.),  etc. 


SECTION  II.— THE   INTESTINAL  AMOEBA. 

Losch  was  the  first  to  record  the  presence  of  an  Amoeba  in  the  stools  of  a 
man  suffering  from  an  ulcerative  affection  of  the  intestine  :  this  organism  he 
designated  Amoeba  coli.  The  same  parasite  was  found  in  the  intestines  of 
persons  suffering  from  dysentery  (Koch,  Hlava)  and  in  dysenteric  abscess  of 
the  liver  (Nasse,  Osier).  Kartulis  has  been  a  strong  advocate  of  the  amoebic 


AMGEB^E  IN  RELATION  TO  DYSENTERY  747 

origin  of  dysentery  and  while  he  has  had  the  support  of  a  number  of  observers 
(Councilman,  Lafleur  and  others),  he  has  been  opposed  by  Tancarat,  Quincke 
and  Roos,  Massintin,  Wilson,  and  others. 

The  following  facts  have  been  urged  against  the  view  that  dysentery  may  be  caused 
by  an  amoeba. 

(1)  Amoebae  are  not  constantly  found  in  persons  suffering  from  dysentery. 
Laveran  only  found  amoebae  in  one  case  of  dysentery  out  of  ten  examined  by 

him,  Krause  and  Pasquale  in  10  out  of  35  cases,  Grasser  in  45  out  of  105  cases,  the 
author  in  2  out  of  12  cases,  Kartulis  in  18  out  of  35  cases,  etc. 

(2)  Amoebae  are  sometimes  found  in  diseases  other  than  dysentery  as  well  as  in 
healthy  persons. 

Quincke  and  Roos  examined  the  stools  of  a  number  of  healthy  persons  after  they 
had  taken  a  dose  of  a  purgative  and  found  amoebae  in  9  out  of  21  cases  :  Grasser, 
Wilson,  Besson  have  also  found  amoebae  in  the  excreta  of  healthy  men.  Sanarelli 
found  amoebae  in  considerable  numbers  in  the  intestines  of  guinea-pigs  which  had 
died  of  enteritis  following  the  ingestion  of  cholera  toxin,  and  in  his  opinion  amoebae 
are  able  to  multiply  in  the  intestine  of  these  animals  in  all  cases  of  toxic  enteritis. 

(3)  Chantemesse,  Shiga,  and  others  have  described  a  bacillus  which  is  obviously 
the  cause  of  a  large  number  of  cases  of  dysentery  (Chap.  XX.). 

Recent  investigations,  however,  have  solved  this  very  vexed  question  of 
the  aetiology  of  dysentery  by  showing  that  the  clinical  term  Dysentery  includes 
two  distinct  diseases. 

(1)  An  acute,  epidemic  disease,  occurring  especially  in  temperate  climates 
and  caused  by  the  bacillus  of  Chantemesse-Shiga. 

(2)  A  chronic,   endemic  disease  prevalent  in  warm  climates,  sometimes 
accompanied  by  abscess  of  the  liver  and  caused  by  an  amoeba. 

It  should  be  mentioned  that  in  the  human  intestine  two  species  of  amoeba  may 
be  encountered  : 

(1)  Amoeba  coli,  non-pathogenic  and  frequently  present  in  healthy  persons; 

(2)  Entamoeba  histolytica,  or  Amoeba  dysenterica,  a  pathogenic  organism  and  the 

cause  of  endemic  dysentery  (Schaudinn,  Jurgens) ; 

and  the  differences  of  opinion  which  are  found  in  the  works  of  some  writers  are  to 
be  explained  on  the  ground  of  a  confusion  of  these  two  species  one  with  another. 

But  though  dysentery  is  generally  due  to  one  or  other  of  the  organisms 
mentioned  there  would  appear  to  be  a  limited  number  of  cases  in  which  the 
symptoms  are  due  to  other  organisms..  Thus,  cases  have  been  recorded  in 
Germany,  Russia  and  warm  countries  in  which  the  infecting  agent  is  a 
parasite  known  as  Balantidium  coli  (vide  infra),  and  other  cases  have  been 
described  in  which  the  following  parasites  appeared  to  be  the  cause  of  the 
symptoms  :  a  Spirillum  (Le  Dantec  at  Bordeaux),  Chilodon  dentatus  (Guiart), 
Trichomonas  intestinalis  (Castellani,  Billet),  the  Hcematozoon  of  Laveran 
(Billet,  Marchoux),  Bilharzia  hcematobia  (Firket,  Letulle,  etc.). 

Microscopical  appearance. 

1.  The  Amoeba  coli  (Entamoeba  coli  of  Schaudinn)  closely  resembles  the 
Amoeba  princeps  and  even  more  closely  Amoeba  pelaqinia  (Mereschowsky), 
another  protozoon  of  very  wide  distribution  outside  the  body. 

In  the  stools  it  occurs  as  a  clear,  slightly  greyish,  rounded  or  elliptical 
mass  of  protoplasm  measuring  15-60//,  in  diameter,  and  as  a  rule  only  pro- 
truding a  single  pseudopodium.  It  consists  of  a  slightly  granular  endosarc 
surrounded  by  a  clear  ectosarc.  The  endosarc  contains  a  rounded  nucleus 
and  one  or  more  highly  contractile  vacuoles,  and  in  addition  there  will  usually 
be  seen  a  number  of  foreign  bodies  (bacteria,  blood  cells,  etc.)  which  have 
been  absorbed  by  the  amoeba.  The  movements  of  the  protozoon  are  very 
limited  and  exceedingly  slow,  so  that  it  does  not  travel  its  own  length  in  a 
minute.  Reproduction  takes  place  by  fission,  the  parasite  dividing  into  two 


748  THE   AMOEBAE 

or  eight  equal  parts.  When  the  conditions  are  unfavourable,  under  the 
influence  of  cold  or  desiccation,  it  becomes  encysted  :  the  cysts  of  Amoeba  coli 
are  large,  and  measure  10//,  or  more. 

2.  Amoeba  (Entamceba)  histolytica  is  found  in  the  stools  of  patients  suffering 
from  ulcerative  dysentery  and  in  tropical  abscess  of  the  liver.  In  the  latter, 
according  to  Rogers,  it  can  only  rarely  be  found  in  the  pus,  but  is  always 
present  in  scrapings  from  the  wall  of  the  abscess.  It  is  distinguished  from 
the  foregoing  species  by  its  more  refractile  ectosarc  sharply  differentiated 
from  the  endosarc,  and  by  its  slightly  elongated  nucleus.  Its  movements 
are  more  rapid  and  the  contour  of  the  parasite  may  be  followed  with  a  camera 
lucida  for  purposes  of  sketching  them  ;  they  are  not  extensive,  being  limited 
to  change  of  shape  rather  than  change  of  position.  The  amoeba  measures  on 
an  average  35/>i  (10-50/x)  in  diameter,  and  it  multiplies  by  binary  fission. 
Cysts  are  formed  on  the  surface  of  the  amoeba  by  a  sort  of  budding  process  ; 
these  cysts  are  much  smaller  (3-6/x  in  diameter)  than  those  of  Amoeba  coli 
(Schaudinn,  Jurgens). 

As  the  causal  agent  of  disease  the  Amoeba  histolytica  passes  through  the 
intestinal  wall,  enters  a  gland  of  Lieberkiihn,  and  reaches  the  sub-mucous 
layer,  where  it  forms  an  abscess. 

Methods  of  detection. 

In  searching  for  intestinal  amoebae  the  examination  should  be  conducted 
on  a  warm  stage  and  the  stools  ought  to  be  examined  immediately  they  are 
passed  and  while  they  are  still  warm.  Pick  up  a  small  flake  of  mucus,  place 
it  on  a  slide  and  compress  it  beneath  a  cover-glass  in  order  to  get  a  thin 
transparent  layer.  If  desirable  the  stools  may  be  diluted  with  a  warm 
solution  of  normal  saline  solution  (O7  per  cent.)  or  with  a  freshly  prepared 
Grassi's  solution,  which  is  perhaps  better. 

Albumin,     •  0'20  gram. 

Sodium  chloride,  1  „ 

Water,         -         -  200        grams. 

The  preparation  should  be  examined  in  the  fresh  state  and  unstained.  A 
few  cysts  will  be  found  in  addition  to  amoebae  and  the  number  of  cysts  will 
increase  as  the  stools  grow  cold.  (Use  a  low  power  objective  to  find  the 
amoebas,  then  turn  on  an  higher  power  dry  lens  to  study  their  structure.) 

The  number  of  amoebae  in  the  stools  varies  considerably  :  occasionally 
they  are  numerous  and  can  easily  be  found,  but  at  other  times  they  may  be 

E resent  only  in  small  numbers  so  that  it  is  difficult  to  detect  them.     In  the 
ttter  cases  the  stools  must  be  examined  on  several  occasions  before  coming 
to  the  conclusion  that  no  amoebae  are  present.     Musgrave  and  Clegg  prescribe 
a  saline  purgative  and  examine  the  liquid  part  of  the  stools,  but  the  following 
method  devised  by  Vincent  gives  better  results. 

Vincent's  method. — Flatten  out  a  flake  of  mucus  as  described  above  and  run  a 
drop  of  a  1  per  cent,  aqueous  solution  of  methylene-blue  under  the  cover-glass.  All 
the  structures  in  the  preparation  with  the  exception  of  the  amoebae  take  up  the 
stain  immediately,  leaving  the  latter  sharply  defined  on  a  blue  background.  As 
the  dye  reaches  the  amoebae  they  throw  out  pseudopodia  and  move  about  actively 
for  a  few  minutes  ;  then  the  movements  become  slower  and  the  parasites  gradually 
take  up  the  dye,  the  nucleus  being  the  last  part  to  stain  ;  finally  the  movements 
cease  altogether  and  the  parasite  dies. 

An  aqueous  solution  of  neutral  red  may  be  used  instead  of  methylene  blue  :  the 
amoebae  then  stain  pink  and  the  other  structures  brick-red. 

Staining  methods. — The  structure  of  the  amoebae  may  be  studied  in  stained 
preparations,  but  before  staining,  the  organisms  must  be  fixed.  Kartulis, 
however,  recommends  staining  dried  preparations  without  fixing  them. 


DETECTION   OF  THE  PARASITES 


749 


(i)  For  an  ex  tempore  preparation  fix  the  amosbse  by  running  a  drop  of  a 
1  per  cent,  solution  of  chromic  acid  under  the  cover-glass  :  then  stain  by 
running  in  a  drop  of  alum-carmine. 

(ii)  For  permanent  preparations  several  methods  have  been  recommended. 


FIG.  353.— Amcfba  coli.     (After  Losch.) 

A.  Bertarelli  recommends  letting  a  drop   of  the  liquid  containing  the 
parasites  dry  on  a  slide,  treating  for  5  minutes  with  absolute  alcohol  very 
lightly  tinted  with  eosin,  washing  with  a  mixture  of  equal  parts  of  alcohol 
and  xylol.  then  with  xylol,  and  mounting  in  balsam. 

B.  Spread  the  preparation  carefully  on  the  slide  and,  before  it  is  dry,  fix 
for  10  minutes  in  an  acetic-perchloride  mixture  (Schaudinn). 

Saturated  aqueous  solution  of  perchloride  of  mercury,      -         100  c.c. 
Absolute  alcohol,  50    ,, 

Glacial  acetic  acid,        -  5  drops. 

Wash  for  10  minutes  in  alcohol  containing  a  little  iodine,  then  in  70  per  cent, 
alcohol  for  30-40  minutes,  and  stain  with  Heidenhain's  or  Grenacher's  haama- 
toxylin  or  with  hsematein  and  eosin. 

C.  Fix  for  10  minutes  in  Flemming's  solution,  wash  in  water,  then  in  alcohol, 
and  stain  with  gentian- violet  or  safranin. 

D.  To  make  out  details  of  structure  fix  in  Schaudinn's  solution  (B,  ante) 
or  in  alcohol  and  stain  by  one  of  the  Romanowsky-Giemsa  methods  or  with 
Laveran's  or  Marino's  stain. 

Sections. — To  prepare  sections  of  the  intestine  or  of  the  wall  of  an  abscess 
proceed  by  one  or  other  of  the  following  methods  : 

A.  Fix  in  Schaudinn's  solution  (B,  ante)  and  stain  with  hsematein  and  eosin. 
The  amoebae  are  bright  pink,  the  nuclei,  violet. 

B.  Fix  as  in  A  and  stain  with  Heidenhain's  iron  hsematoxylin.     The  proto- 
plasm of  the  parasite  is  hardly  stained  at  all  and  the  nucleus  is  outlined  by 
a  blue-black  line. 

C.  Mallory  and  Wright's  method. — Fix  in  alcohol.     Stain  for  5  minutes  in 
a  saturated  aqueous  solution  of  thionin  :  differentiate  in  a  1  per  cent,  solution 
of  oxalic  acid  for  30-60  seconds,  watching  the  preparation  under  the  micro- 
scope and  stopping  the  decolourization  as  soon  as  the  nucleus  assumes  a 
violet  tint :   wash  :   dry  :   mount. 


750 


THE   AMOEBAE 


The   protoplasm   is   streaked   with    blue    and    the    nucleus    is    red    or 
violet. 

D.  Borrel's  method. — (vide  Coccidia,  Chap.  LVII.) 


Cultivation. 

It  has  not  yet  been  definitely  established  that  cultures  of  the  Amoeba  histo- 
lytica  have  been  grown.  Lesage,  who  has  obtained  cultures  with  which  he 
has  caused  dysentery  in  young  cats,  is  of  opinion  that  these  cultures  owe 
their  pathogenic  properties  not  to  the  amoebse  which  have  multiplied  and 
are  probably  saprophytic  species,  but  to  the  Amoeba  histolytica,  which  has 
not  multiplied  but  has  retained  its  vitality.  In  any  case  cultures  of  intestinal 
amoeba?  can  only  be  obtained  by  growing  them  symbiotically  with  bacteria 

and  it  would  seem  that  they  require 
living  bacteria  for  food  (Frosch. 
Mouton,  Musgrave  and  Clegg,  and 
others).  The  medium  should  be 
either  a  very  feebly  nutritive  agar 
prepared  with  agar  which  has  been 
washed  for  a  long  time  (Lesage)  or 
a  similar  medium  containing  a  very 
small  quantity  (O03-0'05  per  cent.) 
of  meat-extract  and  made  alkaline. 
Kartulis  obtained  cultures  by  sowing 
small  amounts  of  stools  in  an  infusion 
of  straw,  but  the  amoebae  only  grew 
provided  that  the  flasks  were  not 
plugged  even  with  wool.  These  experi- 
ments however,  as  Schubert  pointed 
out,  are  valueless  as  evidence  that  the 
amoeba  can  be  cultivated  outside  the 
body  because  the  amoebae  found  in  the 
straw  infusion  were  simply  impurities 
from  the  dust  in  the  air. 

Musgrave  and  Clegg  isolated  in- 
testinal amoebae  by  sowing  stools  on 
agar  plates  previously  sown  with  bacteria,  for  preference  bacteria  isolated  from 
the  intestine  of  the  patient.  These  amoebae,  which  Musgrave  and  Clegg 
found  in  water,  in  soil  and  on  vegetables  in  the  neighbourhood  of  Marseilles, 
would  appear  to  be  saprophytic  species.  Musgrave  and  Clegg  are  of  opinion 
that  all  intestinal  amoebae  may  become  pathogenic. 

Lesage  obtained  cultures  of  amoebae  from  7  out  of  30  cases  of  tropical  dysentery. 
He  sowed  a  little  of  the  intestinal  mucus  either  fresh  or  after  it  had  been  dried 
(encysted  amoebae)  on  the  surface  of  agar  plates.  After  incubating  for  a  few  days 
at  18°-25°  C.  small  amoebae  were  found  at  the  points  where  the  material  was  sown 
and  were  transferred  to  a  fresh  agar  plate  in  such  a  manner  as  to  be  a  little  distance 
away  from  a  colony  of  a  paracolon  bacillus.  Sub-cultures  were  again  made  as 
soon  as  the  amoebae  reached  the  colony  of  bacteria  and  after  sub-cultivating  in 
this  fashion  several  times  a  mixed  culture  of  amoebae  and  paracolon  bacilli  was 
obtained.  Cultures  obtained  in  this  way  are  very  long-lived  :  Lesage  kept  his 
amoebae  in  culture  for  2  years  and  sub-cultured  them  66  times  ;  the  amoeba  lives 
from  4^5  months  in  one  culture  while  the  cyst  retains  its  vitality  for  at  least  6—8 
months. 

More  recently  Lesage  has  found  that  his  cultures  were  merely  cultures  of  sapro- 
phytic amoebae  and  this  would  also  appear  to  be  the  case  with  the  cultures  he 
obtained  by  sowing  dysenteric  stools  on  leucocytes  of  guinea-pigs,  rabbits,  cats 
and  man. 


FIG.  354. — Amoeba  histolytica  in  recent  stools. 
The  figures  represent  the  different  forms  assumed 
by  the  parasite  when  observed  every  15  seconds. 
The  endosarc  contains  a  nucleus  and  three  in- 
gested red  cells.  (After  Jiirgens.) 


EXPERIMENTAL   INFECTION  751 

Experimental  infection. 

It  is  now  established  that  the  introduction  of  the  Amoeba  kistolytica  into 
the  alimentary  canal  of  man,  monkeys  and  young  cats  reproduces  the  lesions 
characteristic  of  dysentery. 

Many  of  the  early  experiments,  carried  out  before  the  amoeba  of  dysentery  was 
identified  and  when  the  fact  that  there  were  several  forms  of  dysentery  was  unrecog- 
nized, only  gave  conflicting  results. 

Losch  injected  recently-passed  dysentery  stools  into  the  alimentary  canals  of 
four  dogs  :  after  the  lapse  of  a  week  amoebae  were  found  in  the  excreta  of  one  only 
of  the  animals :  this  animal  remained  in  apparently  good  health,  but  when  it  was 
killed  on  the  eighteenth  day  the  rectal  mucous  membrane  was  found  to  be  inflamed 
and  ulcerated  in  places  and  the  amoebae  had  multiplied  at  the  site  of  the  ulcers. 

Kovacz  produced  a  blood-stained  diarrhoea  in  a  cat  by  inoculating  it  in  the  rectum 
with  dysenteric  stools. — Kartulis  obtained  a  similar  result  with  one  of  his  straw 
infusion  cultures. — Zancarol  produced  dysentery  in  a  cat  by  inoculating  stools 
containing  amoebae  into  the  rectum  ;  but  he  obtained  the  same  result  with  pus  from 
an  abscess  of  the  liver  which  only  contained  Streptococci  and  also  with  pure  cultures 
of  Streptococci. 

Moreover,  cats  often  suffer  from  an  ulcerative  colitis  resembling  dysentery  (Gasser) 
and  dogs  are  liable  to  a  similar  disease.  The  author  saw  several  dogs  in  Tunis 
affected  with  this  form  of  colitis  and  failed  to  find  amoebae  in  the  dejecta.  In  the 
cat  rectal  injection  of  irritant  substances  and  especially  of  sterilized  soil  produces 
ulceration  of  the  colon. 

Kartulis  infected  a  cat  with  amoebic  dysentery  by  inoculating  it  per  rectum 
with  stools  from  a  patient  suffering  from  amoebic  dysentery.  The  same 
result  can  be  obtained  by  inoculating  pus  from  an  abscess  of  the  liver  con- 
taining amoebae  in  pure  culture  (Kartulis,  Krause)  and  by  operating  in  a 
similar  manner  an  amoebic  dysentery  which  is  almost  always  fatal  can  be 
set  up  in  young  dogs  (Hlava,  Kartulis,  F.  Harris). 

Lesage  inoculated  0*5  c.c.  of  recent  dysenteric  stools  or  pus  freshly  taken 
from  an  abscess  of  the  liver  and  containing  living  amoeba?  into  the  rectum  of 
young  cats.  A  certain  proportion  of  the  animals  suffered  from  symptoms  of 
amoebic  dysentery  with  blood-stained  mucus  in  the  stools  and  died  in  about 
12  days  or  a  fortnight.  Post  mortem,  lesions  characteristic  of  dysentery  were 
found  (thickening,  ulceration  and  necrosis  of  the  mucous  membrane  of  the 
large  intestine).  The  parasites  may  enter  the  blood-stream. 

Young  cats  can  also  be  infected  by  feeding  them  (or  by  means  of  an  ceso- 
phageal  catheter)  with  minced  meat  mixed  with  infected  stools  :  to  produce 
infection  the  stools  must  contain  encysted  amoebae  (desiccated  stools  or  stools 
which  have  been  kept  for  a  few  hours  in  a  moist  chamber). 

Lesage  has  also  infected  young  cats  with  his  cultures  of  amoebae  (vide 
ante). 

Musgrave  and  Clegg  by  feeding  monkeys  (Macacus  cynomolgus  and  M. 
pTiilippinensis]  with  cultures  of  amoebae,  or  by  introducing  the  cultures  into 
their  stomachs,  set  up  a  typical  dysentery  with  haemorrhagic  catarrh  and 
occasionally  small  ulcers  of  the  colon. — A  man  who  had  swallowed  three 
gelatin  capsules  containing  a  three-week-old  culture  of  an  intestinal  amoeba 
together  with  an  harmless  bacillus  suffered  at  first  from  diarrhoea  with 
amoebae  in  the  stools  (twelfth  day)  and  later  from  tenesmus  and  blood- 
stained stools  (twentieth  day). 

Agglutination. — The  blood  of  persons  affected  with  amoebic  dysentery  does 
not  agglutinate  the  bacillus  of  Shiga. 


CHAPTER  LVI. 

Section  I. — The  Microsporidia. 

1.  Nosema  bombycis,  p.  752.     2.  Nosema  apis,  p.  753. 
Section  II. — The  Myxosporidia,  p.  754. 
Section  III. — The  Sarcosporidia,  p.  756. 
Section  IV. — The  Haplosporidia,  p.  759. 

SECTION  I.— THE  MICROSPORIDIA. 

No  Microsporidium  is  known  to  infect  man  but  one  species,  Nosema  bombycis, 
is  the  cause  of  the  silkworm  disease  known  as  pebrine  and  another  species, 
Nosema  apis,  is  the  cause  of  Microsporidiosis  of  bees — the  "  Isle  of  Wight 
disease." 

1.  Nosema  bombycis. 
Synonyms. — Microsporidium  bombycis  :    [Glugea  bombycis]. 

Cornalia  was  the  first  to  observe  the  presence  of  bright  oval  corpuscles  in 
silkworms  affected  with  pebrine.  These  corpuscles  commonly  described  as 
the  corpuscles  of  Cornalia  and  which  have  obtained  so  considerable  a  notoriety 
since  the  investigations  of  Pasteur  and  Balbiani  represent  a  stage  in  the 
life  history  of  the  parasite  which  is  the  cause  of  the  disease. 

The  spore  of  Nosema  bombycis  is  a  small  oval  or  pyriform-shaped  parasite 
measuring  4  x  2/*  surrounded  by  a  spore-membrane  and  containing  at  one 
end  a  single  polar  capsule  in  which  is  hidden  a  spirally-twisted  filament  which 
can  only  with  difficulty  be  demonstrated  (Thelohan). 

For  the  purpose  of  studying  the  development  of  Microsporidium  bombycis  Balbiani 
suggests  the  following  experiment : — Feed  a  number  of  young  silkworms  not  more 
than  a  few  mm.  long  on  mulberry  leaves  which  have  been  washed  over  with  an 
emulsion  made  by  rubbing  up  an  infected  silkworm  moth  with  a  little  water.  In  a 
few  days  the  silkworms  will  be  infected  and  the  spores  will  be  scattered  along  the 
lumen  of  the  alimentary  canal.  The  spores  make  their  way  into  the  wall  of  the  gut 
and  give  origin  to  small  trophozoites  of  variable  size  elongated  in  the  direction  of 
the  longitudinal  muscular  coat. 

Life  history. — [The  development  of  Nosema  bombycis l  in  the  silkworm  begins  as 
small  uninucleate  amcebulse  which  are  in  the  first  instance  found  free  in  the  digestive 
tract  and  later  in  the  lymph  channels.  The  amcebulse  multiply  by  fission,  wander 
all  over  the  body  (planonts)  and  penetrate  cells  where  they  grow,  assume  an  oval 
or  spherical  form  and  become  meronts  or  schizonts.  The  meronts  multiply  by 
binary  or  multiple  fission  until  they  have  filled  and  exhausted  the  host  cell.  They 
do  not  however  pass  to  other  cells.  The  multiplication  of  the  meronts  may  be 
very  similar  in  appearance  to  yeast  cells  and  give  rise  to  chains  of  cells.  When 
the  host  cell  is  used  up  the  meronts  do  not  multiply  further  but  produce  a  final 
generation  of  uninucleate  cells  which  as  sporonts  give  rise  to  a  single^  spore. 

t1  Vide  Minchin,  E.  A.     An  Introduction  to  the  Study  of  the  Protozoa.     London,  1912.] 


THE   MICROSPORIDIA  753 

[The  nucleus  of  the  sporont  (sporoblast)  buds  off  three  small  nuclei,  two  of  which 
form  the  sporocyst  and  the  third  is  concerned  with  the  polar  capsule.  The  principal 
nucleus  remains  as  the  nucleus  of  the  amosbula.  The  protoplasm  probably  also 
divides.  The  sporocyst  when  formed  is  a  tough  capsule  which  though  produced 
by  two  cells  does  not  show  any  indication  of  its  two-fold  origin.  The  spore  is 
egg-shaped  the  anterior  end  being  the  narrower.  The  single  polar  capsule  is  of 
relatively  large  size  and  contains  a  very  long  filament.  The  amcebula  occupies 
the  middle  of  the  spore  and  appears  to  encircle  the  axial  polar  capsule.  The  amcebula 
has  at  first  a  single  nucleus  which  subsequently  divides  into  two  then  into  four. 

[When  the  spore  germinates  in  the  intestine  of  a  new  host  the  polar  filament  is 
extruded  and  the  amcebula  escapes  by  the  pore  at  the  anterior  end.  The  amcebula 
emerges  from  the  spore  with  two  nuclei  leaving  the  other  two  in  the  sporocyst.  The 
two  nuclei  fuse  to  form  a  synkaryon  and  the  now  uninucleate  amoebula  initiates  the 
generation  of  planonts  (Stempell). 

[Hereditary  infection  is  effected  by  the  penetration  of  the  parasite  into  the  ovary 
and  the  formation  of  spores  within  the  ovum  itself.  Hence  not  only  may  the  silk- 
worm be  infected  by  ingesting  spores  of  the  parasite  but  the  newly- hatched  silkworms 
may  already  be  infected.  This  transmission  of  infection  through  the  egg  is  with 
the  possible  exception  of  the  parasite  of  Texas  fever  unique  among  the  Sporozoa.  ] 


FIG.  355.— Nosema  bombycis.  B,  masses  of  Microsporidia  in  a  follicle  of 
the  testis  of  a  silkworm :  S,  mature  spores  :  S'.  immature  spores.  (After 
Balbiani.) 

The  detection  of  the  parasites. — The  Microsporidia  are  highly  resistant  to 
the  action  of  chemical  reagents,  and  can  be  best  seen  in  the  fresh  state, 
unstained,  with  an  high  power  dry  objective.  They  may  be  stained  by 
Vlacovich's  method  :  treat  with  a  32  per  cent,  solution  of  potash  for  48  hours, 
then  with  Gram's  solution,  and  examine  in  a  drop  of  glacial  acetic  acid  :  the 
parasites  are  stained  violet. 

2.  Nosema  apis. 

[An  epizootic  disease  of  bees1  which  on  account  of  the  ravages  it  has 
caused  during  the  past  few  years  among  the  bees  in  the  Isle  of  Wight  has 
popularly  become  known  as  the  Isle  of  Wight  disease  has  been  shown  by 
Fantham  and  Porter  to  be  due  a  microsporidian  parasite  of  the  genus  Nosema 
— Nosema  apis.  The  disease  however  is  widely  spread  and  is  more  suitably 
described  as  Microsporidiosis  of  bees. 

[The  symptoms  of  bee  disease  which  however  seem  to  be  subject  to  considerable 
variation  are  thus  described  by  Virgil:  2 

"  Si  vero,  quoniam  casus  apibus  quoque  nostros 
Vita  tulit,  tristi  languebunt  corpora  morbo — 
Quod  jam  non  dubiis  poteris  cognoscere  signis : 
Continue  est  aegris  alius  color;  horrida  voltum 
Deformat  macies ;  turn  corpora  luce  carentum 
Exportant  tectis  et  tristia  funera  ducunt ; 

f1  See  Supplement  No.  8  to  The  Journal  of  the  Board  of  Agriculture,  Vol.  xix.  No.  2. 
May  1912.] 

[2P.  Vergili  Maronia,  Georgicon,  Liber  Quartus.] 

SB 


754  THE   MICROSPORIDIA 

Aut  illae  pedibus  connexae  ad  limina  pendent, 
Aut  intus  clausis  cunctantur  in  aedibus,  omnes 
Ignavaeque  fame  et  contracto  frigore  pigrae." 

[Infection  takes  place  by  the  contaminative  method  and  may  be  trans- 
mitted through  the  agency  of  infected  foods  or  of.  living  infected  bees. 
Foraging  bees  infected  by  ingesting  food  containing  the  spore  and  "  parasite- 
carriers  "  are  the  most  important  agents  of  infection  (Graham  Smith  and 
Bullamore). 

[The  main  alimentary  tract  of  the  bee,  particularly  the  chyle  stomach 
and  intestine,  are  the  chief  parts  infected.  The  gut  diverticula  appear  to 
be  free  from  parasites,  but  as  the  Nosema  may  be  found  in  the  hsemocoelic 
fluid,  the  parasite  may  occasionally  invade  other  organs  (Fantham  and 
Porter).  It  is  at  present  doubtful  whether  hereditary  infection  occurs  as 
is  the  case  with  Nosema  bombycis  in  silkworms  (vide  supra). 

[The  spores  of  Nosema  apis  occur  as  oval,  highly  refractile  bodies  2— 4/x 
in  diameter  and  4-6/*  in  length.  Frequently  they  are  found  lying  between 
the  cells  of  the  gut  wall  and  in  the  earlier  stages  these  cells  are  distended  with 
the  parasites. 

[Technique. — For  microscopical  examination  small  portions  from  different  parts 
of  the  alimentary  canal  should  be  teased  out  in  a  drop  of  water  and  mounted  under 
a  cover-glass.  As  fixatives,  osmic  acid  vapour  followed  by  absolute  alcohol,  or 
acetic-perchloride  solution  may  be  used.  The  most  useful  stains  are  Giemsa's 
solution  or  haematoxylin.  Sections  give  very  disappointing  results.  For  the 
extrusion  of  the  polar  filament  treatment  with  iodine  water  or  dilute  acetic  acid 
are  recommended. 

[Life  history. — Spores  of  the  parasite,  swallowed  with  food  or  drink  by  the  bee, 
give  rise  each  to  an  amoeboid  parasite  or  planont  which  either  enters  an  epithelial 
cell  of  the  gut  or  reaches  the  hsemocoele.  In  either  case  it  becomes  rounded,  feeds, 
grows  and  then  commences  to  multiply.  The  meront,  as  the  parasite  is  described 
at  this  stage,  divides  by  binary  fission  producing  clusters  or  chains,  each  daughter 
meront  being  ultimately  uninucleate. 

[The  multiplicative  stage  is  followed  by  the  second  or  propagative  stage — sporo- 
gony.  The  full  grown  meront  becomes  the  sporont  or  pansporoblast  which  undergoes 
complicated  nuclear  changes  whereby  five  nuclei  are  ultimately  produced.  The 
sporoblast  forms  two  vacuoles,  an  anterior  one  called  the  polar  capsule  and  a  posterior 
vacuole  in  which  the  polar  filament  is  coiled.  The  secretion  of  the  sporocyst  con- 
verts the  sporoblast  into  the  spore  (Fantham).  ] 

Among  other  species  of  Microsporidia  the  following  may  be  mentioned  : — Nosema 
ovoideum,  a  parasite  of  Motella  tricirrata  and  of  Cepola  rubescens  :  Nosema  bryzotdes, 
found  in  certain  Bryozoa.  Lutz  and  Splendore  have  described  several  species  of 
Microsporidia  in  the  Lepidoptera,  in  different  insects  and  in  a  Cyprinodont  fish. 
Simond  has  found  a  Nosema  in  a  mosquito  (Stegomyia  fasciata) :  this  parasite  seems 
to  be  identical  with  Myxococcidium  stegomyice,  erroneously  described  by  Beyer  and 
by  Parker  and  Pothier  as  the  cause  of  yellow  fever. 


SECTION  II.— THE  MYXOSPORIDIA. 

The  Myxosporidia  are  found  as  parasites  in  fish,  reptiles,  arthropods,  etc. 
They  inhabit  the  skin,  gills  and  internal  organs  :  the  nervous  system  appears 
to  be  the  only  part  of  the  body  never  infected. 

Jaboulay  is  of  opinion  that  cancer  in  man  is  due  to  a  Myxosporidium,  the 
source  of  infection  being  salads,  molluscs,  fish  or  unsuitable  drinking  water. 

Detection  of  Myxosporidia  in  fish.— The  parasites  should  be  looked  for  in 
the  small  prominent  pustules  which  develop  on  the  integuments,  in  the  blood 
cysts  which  form  on  all  the  branches  of  the  splenic  artery  (in  tench),  in  the 


THE  MYXOSPORIDIA 


755 


Myxosporidium 
Cyst  developed 


cysts  on  the  gill-slits,  urinary  bladder  and  swim-bladder,  in  the  spleen,  liver, 
etc. 

If  a  small  cyst — 2-6  mm.  long — be  transferred  to  a  slide  and  examined 
under  the  microscope  it  will  appear  in  the  fresh  unstained  condition  to  consist 
of  an  enveloping  membrane  and  its  contents.  The 
envelope  is  tough,  thick,  and  amorphous,  while  the 
contents  consist  of  a  more  or  less  liquid  material 
(stained  with  hsematoidin  in  arterial  cysts)  contain- 
ing various  granules,  as  well  as  parasites  in  different 
stages  of  development. 

Sections  of  the  internal  organs  (liver,  kidney,  etc.) 
should  be  fixed  in  osmic  acid  or  perchloride  of 
mercury  and  stained  with  safranin  or  gentian- violet 
and  eosin  (Wosielewski)  :  carbol-thionin  is  equally 
useful. 

Morphology. — The  parasites  are  often  rounded 
and  vary  considerably  in  size  (65-300/x)  :  their 
protoplasm  is  finely  granular.  When  examined  in 

fish  urine  they  exhibit  very  slow  movements ;  these   in  the  wail  of  the  mesenteric 
are  not  apparent  when  the  parasites  are  examined 
in  water. 

In  the  parasites  as  just  described  there  appear  at  a  given  moment  small 
rounded  structures  containing  one  or  two  nuclei ;  these  constitute  the  primitive 
spheres  in  which  the  spores  are  formed  (Laveran).  Each  sphere  gives  rise  to 
two  spores  and  some  fatty  granules  which  stain  with  osmic  acid.  The  structure 
of  the  spores  is  complicated  and  varies  in  the  different  species  ;  in  size  they 
vary  from  8  to  36/*.  A  spore  consists  of  an  enveloping  membrane  and  its 

contents  :  the  membrane  of  the  en- 
velope is  formed  of  two  transparent, 
homogeneous  valves  applied  one  to 
another  like  the  two  halves  of  a 
walnut-shell.  At  one  of  the  poles  of 
the  spore  there  appear  one  to  four 
vesicles  or  polar  capsules,  which  stain 
with  methylene  blue,  thionin  or  safra- 
nin. These  polar  capsules  each  elon- 
gate into  a  small  canal  and  become 
attached  to  the  wall  of  the  spore  at 
the  pole,  where  a  very  fine  opening 
communicating  with  the  exterior  is 
formed. 

Each  polar  capsule  contains  a 
spirally- twisted  filament  which  is  very 
difficult  to  see  under  natural  con- 
ditions, but  if  the  preparation  be 
treated  with  a  drop  of  glycerin  or 
potash  solution  the  filaments  suddenly 
unroll  themselves  and  project  from 
the  spore;  these  filaments  are  occa- 
sionally very  well  developed  and  may  be  eight  to  ten  times  the  length  of 
the  spore  (fig.  357).  Besides  the  polar  capsules,  the  spore  contains  an 
homogeneous  protoplasm  and  a  centrally-situated  nucleus  which  can  be 
stained  with  safranin. 

The  spore  constitutes  the  means  by  which  the  parasite  is  conveyed  from 


FIG.  357. — Myxosporidium  of  the  tench.  Mal- 
pighian  corpuscle  containing  a  Myxosporidium. 
Various  forms  of  spores  contained  in  the  corpuscle : 
above,  immature  amoaboid  spores  ;  to  the  right, 
immature  spores.  (After  Balbiani.) 


756  THE   SARCOSPORIDIA 

one  individual  to  another.  Spore  formation  represents  the  sexual  (sporogonic) 
method  of  reproduction. 

According  to  Doflein  and  Laveran  multiplication  of  the  Myxosporidia 
within  the  host  takes  place  by  division  into  two  (equal  or  unequal)  parts 
(schizogony).  The  investigations  of  Thelohan  and  Hofer  on  the  Myxosporidia 
of  fish  and  those  of  Laveran  on  Myxidium  danilewskyi  (a  parasite  of  Cistudo 
europcea)  tend  to  prove  that  infection  takes  place  via  the  alimentary  canal. 

Of  the  numerous  Myxosporidia  which  have  been  described  in  cold-blooded  verte- 
brates the  following  may  be  mentioned  :  Myxidium  danilewskyi,  studied  by  Laveran 
in  a  tortoise  (Cistudo  europcea)  :  M.  Ueberkiihni  ;  Myxobolus  butschli  (in  fresh- water 
fish) ;  M .  cerebralis,  described  by  Hofer  as  the  probable  cause  of  a  disease  of  certain 
trout ;  M .  lintoni  (in  Cyprinodon  variegatus)  ;  M.  cyprini  which  is  the  cause  of 
"  Pockenkrankheit "  of  carp  (Tedeschi) ;  Leptofheca  agilis  (of  salt-water  fish) ;  Cera- 
tomyxa  appendiculata  (a  parasite  of  Lophius  fiscatorius) ;  C.  linospora,  C.  incequcdis 
(of  salt-water  fish). 

SECTION  III.— THE  SARCOSPORIDIA. 

The  Sarcosporidia  are  found  as  parasites  in  the  muscles  (striated  and  non- 
striated)  and  sometimes  in  the  connective  tissues  of  the  mammalia  (mice, 
rats,  monkeys,  pigs,  cattle,  horses,  sheep)  and  birds. 

These  protozoa  are  very  seldom  found  as  human  parasites.  On  two  occa- 
sions Sarcocystis  tenella,  a  very  common  parasite  of  the  sheep,  has  been  found 
in  man  and  on  each  occasion  by  chance — -once  by  Baraban  and  Saint  Remy 
in  the  vocal  cords  of  an  executed  criminal  and  on  the  other  occasion  by  Hoche 
in  the  muscles  of  a  person  who  had  died  of  tuberculosis.  Kartulis  found  a 
sarcosporidium  (Sarcocystis  immitis)  in  the  liver  and  muscles  of  a  Soudanese. 
The  parasites  found  by  Hadden,  Koch,  Klebs  and  Eve  in  the  kidney,  and  by 
Rosenberg  in  the  muscle  of  the  mitral  valve  of  a  woman,  are  [by  some]  con- 
sidered to  be  other  instances  of  sarcosporosis  in  man.  Vuillemin 
thinks  that  systematic  investigation  would  show  Sarcocystis 
tenella  to  be  a  much  more  common  parasite  in  man  than  is 
generally  believed.  A  sarcosporidium  which  is  parasitic  in  elks, 
caribous  and  deer  is  also  said  to  be  capable  of  infecting  man  (H. 
Brooks). 

The  means  by  which  the  Sarcosporidia  are  transmitted  from  one 
host  to  another  are  still  very  imperfectly  understood  but  it  is 
probable  that  infection  takes  place  through  the  alimentary  canal  and 
may  be  the  result  of  the  ingestion  of  infected  meat.  Grey  mice 
have  been  infected  by  feeding  them  on  the  muscles  of  other  mice 
infected  with  Sarcosporidia  (Smith,  Koch)  and  in  these  cases  there  was 
FIG.  358.  —  a  minimum  incubation  period  of  45  days  before  the  parasite  appeared 
m  *^e  tissues-  Negri  has  infected  guinea-pigs  by  feeding  them  with 
(After  Laveran  Sarcocystis  muris.  [Kartulis'  case  (ante)  affords  some  support  to  the 
capsu^e^31^'  nu-  view  that.  t.he  alimentary  canal  is  the  channel  of  infection,  because 
cleus  surrounded  Sarcosporidia  were  found  in  small  numbers  in  that  situation  and  they 
by  granules.  may  quite  conceivably  have  passed  through  the  intestinal  wall  into 

the  branches  of  the  portal  vein  and  so  reached  the  liver  (Guiart).  ] 
Morphology. — According  to  Laveran  and  Mesnil  the  Sarcosporidia  should 
all  be  grouped  in  one  genus  (Sarcocystis)  and  in  their  view  there  is  no  sufficient 
ground  for  dividing  them,  as  was  proposed  by  Blanchard,  into  two  families 
according  as  to  whether  they  are  found  in  the  muscles  [Miescheria]  or  connec- 
tive tissues  [Balbiania]. 

These  two  families  were  until  recently  recognized  as  comprising  three  genera 
which  were  differentiated  by  the  thickness  of  the  enveloping  membrane  or  cuticle 
of  the  parasite.  The  genus  Miescheria  included  intra-muscular  species  surrounded 


THE   SARCOSPORIDIA  757 

by  a  thin  membrane  and  the  genus  Sarcocystis  the  intra-muscular  species  which 
had  a  thick  capsule  penetrated  by  fine  canaliculi.  The  genus  Balbiania  comprised 
the  parasites  found  in  the  connective  tissues  and  these  had  a  thin  cuticle.  These 
so-called  genera  are,  however,  merely  stages  in  the  life  history  of  the  same  parasite 
(Laveran  and  Mesnil). 


FIG.  359.— Section  through  a  muscle  infected  with  Sarcocystis  tendla. 

Sarcosporidia  can  be  most  conveniently  studied  in  the  muscles  of  sheep 
and  pigs  in  which  they  are  sometimes  found  in  enormous  numbers — chiefly 
in  the  oesophagus,  tongue,  psoas  and  diaphragm. 

If  a  portion  of  an  infected  muscle  be  examined  under  the  microscope  the 
parasite  will  appear  as  a  whitish  fusiform  or  spindle-shaped  body  measuring 
1-5  mm.  long  lodged  in  the  muscular  fibre,  its  long  axis  running  in  the  long 
axis  of  the  muscle.  These  elongated  spindles  represent  Miescher's  or  Rainey's 
tubes  [so  called  after  Miescher,  who  discovered  sarcosporidia  in  the  muscles 
of  mice  and  after  Rainey,  who  found  similar  parasites  some  years  later  in 
pigs'  muscles].  The  membrane  enclosing  these  tubes  is  at  first  a  fine,  struc- 
tureless cuticle  but  before  long  it  thickens  and  becomes  channelled  by  numerous 
very  fine  canaliculi  (arranged  for  the  most  part  transversely  to  the  long  axis 
of  the  parasite,  but  towards  the  extremities  directed  obliquely  and  at  the  tip 
lying  in  the  direction  of  the  long  axis).  The  extreme  tenuity  of  the  membrane 
(ca.  1/x)  can  be  demonstrated  in  sections  of  sarcosporidia  (Laveran  and  Mesnil). 

As  the  parasite  grows  it  gradually  distends  and  destroys  the  muscular  fibre 
in  which  it  is  parasitic,  until  finally  it  is  surrounded  merely  by  the  sarcolemma 
and  sarcoplasma  and  drops  out  in  most  cases  into  the  connective  tissue. 
This  then  is  the  way  in  which  the  intra-muscular  parasite  (Sarcocystis) 
becomes  the  connective  tissue  parasite  (Balbiania). 

While  the  parasite  is  growing  at  the  expense  of  the  muscular  fibre  in  which 
it  has  taken  up  its  abode  the  nucleus  divides  into  a  number  of  secondary 
nuclei  around  which  the  protoplasm  segregates,  thus  forming  a  number  of 
sporoblasts  which  in  turn  segment,  giving  origin  to  the  so-called  falciform 
corpuscles  or  sporozoites. 

On  leaving  the  muscle  arid  passing  into  the  connective  tissue  the  spindle- 
shaped  parasite  is  surrounded  by  a  second  capsule  derived  from  the  muscular 
fibre  (Laveran  and  Mesnil).'  It  now  changes  its  shape,  becomes  rounded, 
and  at  the  same  time  increases  in  size  (up  to  1  cm.)  by  an  increase  in  the 
number  of  sporozoites,  while  its  cuticle  or  membrane  is  so  distended  as  to 


758  THE   SARCOSPORIDIA 

be  again  thin  and  structureless.  The  parasite  then  bursts  its  capsules,  the 
sporozoites  are  set  free  and  a  further  infection  of  the  host  takes  place. 

Infection  of  new  hosts  appears  to  be  effected  by  the  curved,  non-motile, 
sausage-shaped  sporozoites  described  by  Laveran  and  Mesnil.  These  sporo- 
zoi'tes  have  rounded  ends,  of  which  one  is  larger  than  the  other  and  contains 
a  large  nucleus  surrounded  by  chromatin  granules  ;  the  other  end  is  some- 
what more  pointed  [and  contains  a  striated  body  which  may  be  the  analogue 
of  the  polar  capsule  of  the  Myxosporidia  (Guiart)].  The  sporozoites  are  very 
fragile  and  can  be  readily  dissociated  by  keeping  them  in  a  moist  chamber 
or  by  treating  them  with  very  dilute  acids  or  alkalis.  They  probably  do  not 
represent  the  form  which  the  parasite  assumes  outside  the  body  (Laveran 
and  Mesnil). 

Principal  species  of  Sarcocystis  : 

Sarcocystis  muris,  found  in  mice  and  rats. 

Sarcocystis  miescheriana,  a  parasite  of  pigs,  but  does  not  seem  to  have  been  found 
in  man. 

Sarcocystis  tenella,  a  common  parasite  of  sheep  and  goats :  this  parasite  has  been 
found  in  man  (ante).  It  would  appear  to  be  related  to  Balbiania  gigantea,  found  in 
the  oesophagus  of  sheep,  and  apparently  a  giant  form  of  S.  tenella. 

Sarcocystis  immitis,  found  by  Kartulis  in  multiple  abscesses  of  the  liver  and 
muscles  of  a  Soudanese. 

Balbiania  mucosa,  found  by  Blanchard  in  the  kangaroo  and  in  the  connective  tissues 
of  a  Soudanese,  and  Balbiania  siamensis,  a  parasite  of  Siamese  buffaloes,  are  very 
closely  allied  to  B.  gigantea  and  are  therefore  included  in  the  genus  Sarcocystis. 

Sarcocystis  blanchardi,  a  parasite  of  European  and  Javanese  buffaloes. 

Toxin. — Pfeiffer  found  that  an  aqueous  extract  of  Sarcosporidia  inoculated 
beneath  the  skin  of  a  rabbit  led  to  a  fall  of  temperature,  diarrhoea,  and  ulti- 
mately to  the  death  of  the  animal.  Laveran  and  Mesnil  repeated  Pfeiffer's 
experiments  and  proved  the  existence  in  the  Sarcosporidium  of  sheep  (S. 
tenella)  of  a  toxin  to  which  they  have  given  the  name  sarcocystine.  Laveran 
and  Mesnil  prepared  both  aqueous  and  glycerin  extracts  of  Sarcosporidia  ; 
the  aqueous  extract  was  found  to  lose  its  toxicity  rapidly,  so  that  in  6  days 
it  was  already  much  less  toxic  than  when  prepared  ;  the  glycerin  extract, 
on  the  other  hand,  which  is  quite  as  toxic  as  the  aqueous  extract,  keeps  much 
better  and  preserves  its  toxicity  unaltered  for  about  a  month. 

Preparation  of  the  toxin. — Enucleate  a  number  of  Sarcosporidia  from  the  oesophagus 
of  a  sheep  and  after  weighing  them,  crush  them  up  in  a  mortar  with  sterile  sand  and 
a  known  volume  of  water  or  glycerin  (according  as  to  whether  an  aqueous  or  glycerin 
extract  is  to  be  prepared) :  filter  the  aqueous  extract  through  a  porcelain  bougie 
and  the  glycerin  extract  through  paper. 

If  Sarcosporidia  be  opened  and  inserted  beneath  the  skin  they  give  rise  to 
the  same  symptoms  as  the  extracts,  but  if  the  cuticle  be  intact  symptoms  of 
intoxication  are  delayed.  Laveran  and  Mesnil  also  prepared  a  highly  toxic 
dry  extract. 

Preparation  of  dry  extract. — A  number  of  Sarcosporidia  are  dried  in  a  desiccator 
over  sulphuric  acid  and  powdered  ;  the  white  powder  constitutes  the  extract  and 
must  be  stored  in  small  sealed  tubes.  One  eg.  of  the  dry  extract  is  equivalent  to 
5  or  6  eg.  of  fresh  Sarcosporidia. 

Action  on  the  lower  animals.— While  very  toxic  for  rabbits  Sarcocystine  is 
almost  without  effect  on  other  animals. 

On  rabbits. — A  weight  of  extract  equivalent  to  1  mg.  of  fresh  Sarcosporidia  inocu- 
lated beneath  the  skin  of  a  rabbit  weighing  1  kg.  leads  in  about  2  or  3  hours  to  an 
attack  of  diarrhoea  accompanied  by  a  fall  of  temperature  to  below  normal :  the 
cholera-like  symptoms  become  rapidly  more  marked,  convulsions  set  in  and  death 
occurs  in  about  5-10  hours.  Smaller  doses  of  toxin  give  rise  to  a  slight  oedema  at 
the  site  of  inoculation,  rise  of  temperature,  and  wasting  ;  diarrhoea  is  a  late  symptom 


THE   HAPLOSPOBIDIA  759 

and  is  accompanied  by  a  slight  fall  of  temperature  below  normal ;  death  occasionally 
takes  place  about  the  twentieth  day.  Post  mortem  examination  reveals  no  lesion 
of  importance. 

Inoculation  of  the  aqueous  extract  into  the  peritoneal  cavity  has  the  same  effect 
as  sub-cutaneous  inoculation.  After  intra-venous  inoculation  symptoms  develop 
rather  more  rapidly.  Inoculation  of  large  doses  into  the  brain  gives  rise  to  the 
same  symptoms  as  the  inoculation  of  similar  doses  beneath  the  skin.  Feeding 
experiments  and  the  injection  of  the  aqueous  extract  into  the  small  intestine  do  not 
prove  fatal. 

Mesnil  extracted  a  similar  sarcocystine  from  Sarcosporidia  found  in  the  oeso- 
phagus of  an  Hungarian  buffalo.  An  emulsion  in  normal  saline  solution  of 
Sarcosporidia  from  a  Llama  inoculated  into  rabbits  gave  rise  to  symptoms 
referable  to  the  nervous  system — ascending  paralysis,  sub-normal  temperature, 
etc.  ;  there  was  no  diarrhoea  (Rievel  and  Behrens). 

Properties  of  sarcocystine. — The  properties  of  sarcocystine  resemble  those 
of  certain  bacterial  toxins.  The  aqueous  extract  loses  its  toxicity  when 
heated  at  100°  C.  for  5  minutes  or  at  85°  C.  for  20  minutes.  The  glycerin 
extract  is  more  resistant  to  the  action  of  heat ;  after  heating  at  85°  C.  for 
30  minutes  it  will  still  prove  fatal  to  rabbits  if  inoculated  in  large  doses. 

By  mixing  an  aqueous  extract  of  sarcocystine  with  Gram's  solution  or  a 
1  in  12  solution  of  hypochlorite  of  sodium  its  toxicity  is  lowered. 

The  toxicity  of  the  extract  is  not  diminished  by  triturating  it  with  rabbits' 
brain  or  muscles,  so  that  the  toxin  is  not  fixed  by  these  tissues. 


SECTION  IV.— THE   HAPLOSPORIDIA. 

Caullery  and  Mesnil  include  under  this  heading  certain  Protozoa  which, 
though  related  to  the  Sarcosporidia  and  the  Microsporidia,  are  characterized 
by  the  absence  of  polar  capsules  in  the  sporozoi'tes. 

O'Kinealy  described  a  vascular  tumour  of  the  nose  in  a  man  in  Calcutta 
which  was  due  to  an  Haplosporidium  (Rhinosporidium  kinealyi).  This 
tumour  contained  encysted  parasites  of  a  spherical  shape. 

The  young  parasites  have  a  granular  protoplasm  surrounded  by  an  hyaline 
membrane  and  containing  several  small  nuclei :  later,  the  enveloping  mem- 
brane thickens  and  the  protoplasm  segments  around  the  nuclei  to  form 
sporoblasts  and  these  in  turn  divide  and  form  sporozoi'tes  which,  being  set  free 
by  rupture  of  the  cyst,  give  rise  to  new  parasites. 


CHAPTER  LVII. 
THE    COCCIDIIDEA. 

Section  I. — The  genus  Coccidium,  p.  760. 

1.  Coccidium  cuniculi,  p.  760. 

Morphology,  p.  761.     Life  history,  p.  762. 

2.  Other  principal  species  of  Coccidia,  p.  764. 
Section  II. — The  genus  Klossia,  p.  765. 

Section  III. — Parasites  in  tumours,  p.  766. 

1.  Coccidia,  p.  766.     2.  Micrococcus  neoformans,  p.  769. 

[THE  Coccidiidea  are  Sporozoa  belonging  to  the  sub-division  Telosporidia — 
parasites  "  in  which  the  reproductive  phase  of  the  life-cycle  is  distinct  from, 
and  follows  after,  the  trophic  phase  "  (Minchin).] 

The  Coccidia  are  found  as  intra-cellular  parasites  both  in  the  Vertebrata 
and  in  the  Invertebrata.3 

The  Coccidia  are  small,  oval  or  spherical,  nucleated  amoeboid  bodies  with 
granular  protoplasm.  Reproduction  takes  place  both  asexually  (schizogony) 
and  sexually  (sporogony).  The  investigations  of  Leger  and  of  Mesnil  have 
shewn  that  the  Coccidia  should  be  classified  with  the  Haematozoa 
(Chap.  LVIIL). 


SECTION  I.— THE   GENUS   COCCIDIUM. 

Coccidium  cuniculi. 

(Coccidium  oviforme.} 

Whitish  or  yellowish  masses  resembling  small  softened  abscesses  and  con- 
taining oval-shaped  structures  similar  to  the  eggs  of  Nematodes  are  frequently 
to  be  found  lodged  in  the  hepatic  canaliculi  or  parenchyma  of  the  livers  of 
rabbits:  these  masses  are  in  reality  Coccidia.  A  Coccidium  consists  of  a 
retractile  enveloping  membrane,  granular  protoplasm,  nucleus  and  nucleolus. 

The  infection  in  the  rabbit  often  resolves  spontaneously  :  the  Coccidia  are  expelled 
as  oocysts  (vide  infra),  and  on  post  mortem  examination  nothing  more  than  cicatricial 
scars  on  the  surface  or  in  the  substance  of  the  liver  are  left  as  an  indication  of  a 
previous  infection.  In  young  rabbits  Coccidia  may  multiply  very  rapidly  (Pfeiffer), 
in  which  case  the  infection  is  scattered  throughout  the  liver ;  the  biliary  canals  are 
dilated  and  the  connective  tissue  hypertrophied,  compressing  the  blood-vessels 

1  Speaking  generally,  the  evolution  of  the  Coccidia  takes  place  within  the  cells  of  the 
animal  infected.  Laveran  and  Mesnil  have,  however,  described  a  Coccidium  in  a  tortoise 
in  which  the  development  was  entirely  extra-cellular  :  this  may  perhaps  also  be  the  case 
with  C.  bigeminum  (vide  infra). 


COCCIDIUM  CUNICULI  761 

and  leading  to  atrophy  of  the  liver  substance.  The  internal  organs  are  wasted 
and  discoloured,  the  blood  is  pale-coloured  and  watery,  and  the  animal  eventually 
dies. 

The  Coccidium  of  the  rabbit  may  infect  man.  Gubler  has  seen  as  many 
as  twenty  purulent  cysts  in  the  human  liver,  of  the  size  of  a  walnut  or  hen's 
egg,  in  which  the  parasites  were  actively  multiplying.  In  a  case  recorded  by 
Silcocks  the  liver,  spleen  and  intestines  were  in- 
fected, the  parasite  being  found  in  all  the  lesions. 

The  Coccidium  described  as  Coccidium  perforans 
or  G.  hominis,  and  found  in  the  epithelial  cells  of 
the  intestine  of  the  rabbit  and  man,  has  now  been 
shown  to  be  the  same  species  as  C.  cuniculi  (Rivolta, 
Metzner).  This  parasite  has  been  found  by  chance 
during  post  mortem  examinations  and  during  ex- 
aminations of  the  stools  for  other  parasites  ;  in 
most  cases  it  did  not  appear  to  be  responsible  for 
any  special  symptoms,  but  Railliet  and  Lucet  have  «T.y  | 
found  it  in  two  cases  associated  with  symptoms  of 
chronic  diarrhoea.  | JX 

Morphology.  FlG  360.— Coccidiosis  in  the  rabbit. 

If  the  contents  of  one  of  the  cysts  be  ex- 
amined under  the  microscope  in  a  drop  of 
water  or  normal  saline  solution  the  parasite  can  be  seen  quite  distinctly. 
The  Coccidia  stain  badly,  so  that  if  a  drop  of  an  aqueous  solution  of  eosin 
be  added  to  the  above  preparation  the  parasites  stand  out  conspicuously 
as  unstained  objects  on  a  pink  background. 

For  studying  the  structure  of  Coccidia,  Pianese  fixes  small  pieces  of  the  liver 
for  36  hours  in  a  mixture  consisting  of : 

10  per  cent,  aqueous  solution  of  cobalt  chloride,       -  20  c.c. 

2  per  cent,  aqueous  solution  of  chromic  acid,  5     ,, 

Formic  acid,         .....  1  drop. 

Bertarelli  advises  fixing  in  a  saturated  aqueous  solution  of  perchloride  of  mercury, 
staining  for  24-48  hours  in  a  dilute  solution  of  Grenacher's  haematoxylin  (1  c.c. 
hsematoxylin  in  200  c.c.  of  water)  and  differentiating  in  acetic-alcohol. 

Borrel  gives  a  method  for  fixing  and  staining  which  is  particularly  useful  for 
studying  sporozoa  in  sections  : — 

Place  very  small  pieces  of  the  tissue  in  the  following  mixture  for  24  hours  in  the 
ice  chest : 

Osmic  acid,  2  grams. 

Platinum  chloride,        -  2       „ 

Chromic  acid,       -  3       ,, 

Acetic  acid,  20       „ 

Distilled  water,    -  -         350 

Wash  in  a  large  quantity  of  water. 
Embed  in  paraffin. 

Stain  thin  sections  for  1  hour  in  the  cold  in  a  saturated  aqueous  solution  of  Magenta 
red  and  differentiate  for  5—10  minutes  in  the  following  solution : 

Saturated  aqueous  solution  of  picric  acid,  1  volume. 

Saturated  aqueous  solution  of  indigo -carmine,  2  volumes. 

Wash  rapidly.  Decolourize  in  absolute  alcohol,  then  hi  clove  oil,  and  leave  the 
section  in  clove  oil  for  some  little  time.  Mount  in  balsam.  The  nuclei  will  be  stained 
red,  the  protoplasm  blue-green,  and  haemoglobin  yellow  or  yellowish  green. 

Coccidium  cuniculi  is  found  in  the  epithelial  cells  lining  the  bile  ducts.  In 
these  cells  the  cysts  which  are  oval  in  shape  measure  about  4(V  long  x  20/A 
broad.  They  are  filled  with  granular  protoplasm  which  soon  retracts  from 
the  wall  and  forms  a  separate  sphere  with  a  centrally  placed  nucleus  :  this 


762 


THE   COCCIDIA 


represents  the  oocyst  stage— the  last  stage  in  the  life  history  of  the  parasite 
in  the  animal  tissues  (fig.  361). 

The  oocysts  then  pass  into  the  intestine  and  are  eventually  excreted  : 
outside  the  body  of  the  host  they  undergo  a  metamorphosis  which  renders 
them  capable  of  setting  up  fresh  infections  in  other  animals  (the  disease  is 
not  directly  contagious,  or  miasmatic  as  older  writers  would  have  termed  it). 


FIG.  361. — Coccidium  cuniculi.     Encysted 
adult  forms.    (After  Blanchard.) 


FIG.  362. — Coccidium  cuniculi.  Extra-cellu- 
lar life  history.  C,  formation  of  sporoblasts ; 
D,  transformation  of  sporoblasts  into  sporo- 
cysts.  (After  Blanchard.) 


Life  history. 
I.  Outside  the  animal  body. 

If  a  number  of  oocysts  be  placed  in  a  drop  of  sterile  water  x  in  a  Koch's  cell 
and  kept  at  a  temperature  of  15°-18°  C.  their  contents  will  be  seen  in  the  course  of 
2  or  3  days  to  divide  into  two,  and  later,  into  four  small  spheres  or  sporoblasts 
(fig.  362).  Each  sporoblast  then  elongates  and  forms  a  sporocyst  or  cytospore,  each 
of  which  in  turn  divides  into  two  nucleated  falciform  corpuscles,  crescent  bodies  or 
sporozo'ites,  and  a  granular  residuum  which  is  not  utilized  (fig.  363). 


FIG.  363. — Coccidium  cuniculi.  E,  an 
isolated  sporocyst ;  s,  s,  sporozo'ites  ;  r,  resi- 
duum ;  F,  an  isolated  sporozo'ite.  (After 
Balbiani.) 


FIG.  364. — Coccidium  cuniculi.  A,  sporpzoiite 
penetrating  an  epithelial  cell ;  B,  formation  of 
the  schizont. 


II.  Infection  of  the  host. 

The  cyst  containing  the  sporocysts  is  highly  resistant  to  external  influences  and 
retains  its  vitality  for  a  long  time.  When  a  rabbit  swallows  such  a  cyst  the  capsule 
is  digested  and  the  sporocysts  are  set  free,  and  these  in  turn  open  and  discharge  the 
sporozoi'tes.  The  sporozoites  are  actively  motile  and  so  are  able  to  pass  into  the 

1  The  following  method  is  recommended  by  Leger  and  by  Laveran  for  studying  the 
extra-corporeal  development  of  coccidia :  Lay  the  material  containing  the  coccidia  on 
small  pieces  of  charcoal  and  place  the  charcoal  in  watch-glasses  containing  a  few  drops  of 
carbolized  water  (to  prevent  the  growth  of  moulds  and  bacteria)  and  keep  the  preparation 
in  a  moist  chamber. 


COCCIDIUM  CUNICULI 


763 


biliary  passages.  On  coming  in  contact  with  an  epithelial  cell  the  sporozoiite  by  its 
pointed  anterior  end  penetrates  and  passes  entirely  into  the  cell,  reaches  the  centre 
of  the  cell-protoplasm  (between  the  nucleus  and  the  free  surface),  loses  its  motility 
and  soon  assumes  a  new  appearance,  the  schizont  (fig.  364),  which  grows  at  the 
expense  of  the  cell  in  which  it  is  living  and  multiplies  by  asexual  division  (schizogony). 

III.  In  the  body  of  the  host. 

A.  Schizogony  (Asexual  reproduction). — The  schizont,  which  has  no  enveloping 
membrane,  grows  and  assumes  a  spherical  form,  while  its  protoplasm  becomes 
hollowed  out  by  large  alveoli  filled  with  a  clear  fluid.  The  nucleus  soon  divides  into 
a  large  number  of  daughter  nuclei  which  pass  towards  the  periphery :  the  proto- 
plasm divides  into  an  equal  number  of  segments  and  accumulates  around  the  nuclei. 
Thus  a  number  of  claviform  corpuscles  are  formed,  arranged  at  first  like  the  quarters 
of  an  orange  but  later  becoming  free  and  exhibiting  movements  similar  to  those  of 
sporozoa.  These  represent  the  merozoites  (fig.  365). 


FIG.     365.  —  Cocddium    cuniculi. 
nucleus  ; 
Simond.) 


Schizogony.     A,    multiplication 
B,  multiplication  of  the  cell  ;    C,  fully-grown  merozoites. 


of    the 
(After 


At  this  moment  the  epithelial  cell  bursts  and  the  merozoites  are  set  free  :  some 
die  in  the  intestine  of  the  rabbit  ;  others  penetrate  fresh  epithelial  cells,  where 
one  of  two  things  may  happen. 

In  some  cases  a  merozoite  entering  a  healthy  cell  loses  its  motility,  becomes 
spherical,  increases  in  size  and  forms  a  true  schizont  and  at  once  begins  to  multiply 
in  the  manner  already/  described.  By  repeated  schizogony  the  parasite  can  multiply 
very  quickly  in  the  rabbit's  tissues  and  this  explains  the  very  rapid  manner  in  which 
infection  sometimes  spreads  through  the  body. 

In  other  cases  a  merozoite,  after  penetrating  an  epithelial  cell,  undergoes  changes 
preparatory  to  a  sexual  mode  of  reproduction. 

B.  Sporogony  (Sexual  reproduction).  —  In  this  case  some  of  the  merozoites  after 
entering  a  cell  of  the  host  are  converted  into  female  cells,  macrogametes,  others  into 
male  cells,  microgametes. 

1.  Macrogametes.  —  The   merozoite  destined  to   become  a  macrogamete  slowly 
increases  in  size,  and  provides  itself  with  reserve  material  in  the  form  of  chromatin 
granules.     The  nucleus  contains  a  karyosome  which  before  long  is  expelled,  and 
the  macrogamete,  elliptical  in  shape,  exhibits  contractile  movements  which  generally 
result  in  its  passing  out  of  the  cell-host  ;   it  then  remains  on  the  surface  of  the  epi- 
thelium where  it  can  be  easily  reached  by  the  microgametes.     The  now  mature 
macrogamete  is  spherical,  non-motile,  and  has  a  sharply  defined  nucleus  ;  the  granu- 
lations pass  to  the  periphery,  fuse,  and  form  an  enveloping  membrane  pierced  at 
one  end  by  an  orifice,  the  micropyle. 

2.  Microgametes.  —  The  merozoite  about  to  be  transformed  into  a  male  cell  has 
no  enveloping  membrane  or  granules  of  reserve  material.     It  grows  rapidly  and 
soon  becomes  converted  into  the  microgametoblast  from  which  the  microgametes  will 
take  origin.     The  nucleus,  which  has  a  large  karyosome,  divides  into  a  number  of 
daughter  nuclei  and  these  range  themselves  round  the  periphery  of  the  organism 
and  around  each  of  them  a  mass  of  hyaline  protoplasm  collects.     The  daughter 
nuclei  soon  become  flattened,  elongated  and  comma-shaped.     These  are  the  micro- 
gametes  ;  they  continue  to  elongate  and  iwoflagella  appear  at  their  anterior  extremity 
(the  point  of  insertion  of  the  flagella  varying  in  different  species)  :   the-  microgametes 
are  motile  and,  becoming  free,  leave  the  microgametoblast,  which  forms  a  residual 
body  and  is  soon  destroyed. 


764 


THE   COCCIDIA 


The  microgamete  bears  a  considerable  resemblance  to  the  spermatozoa  of  higher 
animals :  it  is  very  small  (6-8/x  long),  actively  motile,  generally  falciform,  with  an 
homogeneous  refractile  body,  and  is  almost  entirely  composed  of  chromatin  surrounded 
by  a  very  thin  layer  of  protoplasm  (fig.  366). 

3.  Fertilization. — The  mature  macrogamete  attracts  the  microgametes  by  chemio- 
tactic  influences :  a  single  microgamete  penetrates  the  female  cell  at  the  micropyle 
and  as  soon  as  it  has  passed  into  the  macrogamete  the  micropyle  closes  behind  it. 
The  male  element  reaches  the  nucleus  of  the  macrogamete  and  fuses  with  it,  forming 


FIG.  366. — Free  microgametes  of  Echinospora. 
(After  Leger.) 


FIG.  367. — Fertilization  in  Coccldium  schubergi. 
(After  Schaudinn.) 


the  oocyst,  and  fertilization  is  now  completed.1  The  oocyst  then  leaves  the  body 
of  its  host,  matures,  and  divides  by  sporogony,  giving  rise  to  sporocysts,  which  will 
infect  new  hosts  (vide  ante).2 


Other  principal  species  of  coccidia. 

The  genus  Coccidium  comprises  about  forty  species  which  are  parasitic  in 
the  mammalia,  birds,  reptiles,  batrachians,  fish,  myriapods  and  insects.  The 
following  species  may  be  mentioned  in  addition  to  those  already  noticed  : 

Coccidium  falciforme,  a  parasite  of  Mus  musculus  ;  C.  avium  (vel  C.  tenellum), 
a  parasite  infecting  fowls,  pheasants,  etc.  ;  C.  salamandrce  ;  C.  pfeifferi,  a 
parasite  of  doves  ;  C.  jalinum,  observed  by  Perron9ito,  Dematteis  and  Borini 
in  a  case  of  chronic  enteritis. 

According  to  Wasiliewski,  C.  bigeminum  (vel  Cystospermium  villorum  intestinalium 
canis)  which  has  its  habitat  in  the  villi  of  the  intestine  of  dogs  and  cats,  is  probably 
the  same  parasite  as  that  found  by  Kjellberg  hi  Berlin  in  the  intestinal  villi  of  a 
man,  and  should  be  classified  with  the  Diplospora,  a  genus  closely  related  to  the 
Coccidia  and  characterized  by  the  fact  that  the  oocyst  produces  two  sporocysts  each 
giving  origin  to  four  sporozoi'tes. 

The  genus  Eimeria  (Schaudinn),  in  which  Blanchard  includes  the  parasite  found 
by  Kiinstler  and  Pitres  in  a  case  of  pleurisy  in  man  (E.  hominis),  should  apparently 
be  classed  among  the  Coccidia. 

Unclassified  Coccidia. — Kartulis  has  recorded  cases  in  which  Coccidia  were  the 
cause  of  tumours  in  muscles.  Londermann  found  brownish-looking  tubercles, 
2-3  mm.  in  diameter,  and  containing  coccidia,  in  the  sigmoid  valves  of  the  aorta 

1  The  fertilized  cell  is  sometimes  known  as  the  zygote. 

2  In  some  species,  the  oocyst  matures  in  the  tissues  of  the  infected  host  and  may  at 
once  infect  new  hosts  without  passing  through  an  extra-cellular  stage.     These  coccidia 
are  directly  contagious  (C.  truncatum  of  the  goose  ;  C.  proprium  of  the  triton). 


THE   GENUS   KLOSSIA 


765 


and  in  the  mitral  valve  of  a  patient  who  had  died  of  anasarca.  Coccidia  have  also 
been  found  in  the  kidneys  (Lindermann) ;  in  the  skin  (Milian,  Cornil  and  Duret) ; 
and  in  the  liver  (Gubler,  Virchow,  etc.). 


\Z 
FIG.  368.— Life  history  of  a  Coccidium.     (Scheme  after  Schaudinn.) 

A.  Schizogony. — 1.  A  sporozoiite  free  in  the  intestine  of  the  host.     2.  Penetra- 
tion into  an  epithelial  cell.     3,  4.  Growth  of  sporozoite  into  trophozolte.    5,  6,  7. 
Stages  in  asexual  multiplication.     8.  Free  merozoftes. 

B.  Sporogony. — 9a.    Undifferentiated   female   cell.     lOa.    Macrogametocyte. 
13a.  Macrqgamete.     A  single  macrogamete  only  is  formed  from  each  female  cell. 
— 9b.  Undifferentiated  male  cell.     lOb.  Microgametocyte.     11, 12.  Formation  of 
several  microgametes  from  a  single  male  cell.     13b.  Microgamete. — 14.  Fer- 
tilization.    15,  16,  17.     Zygote.     18,  19.  Formation  of  spores.     20.  Formation 
of  sporozoites  within  the  spores.     21.  Sporozoltes  released  in  intestine  of  host 
(From  Mense's  "  Handbuch  der  Tropenkrankheiten.") 


SECTION  II.— THE   GENUS  KLOSSIA. 

Klossia  Jielicina  is  an  excellent  species  for  the  study  of  the  Coccidia  and  for 
this  reason  a  short  description  of  it  is  given  here.  The  parasite  lives  and  is 
almost  always  to  be  found  in  the  tissues  of  Helix  hortensis.  Its  life  history 
has  been  described  by  Laveran. 

Salomonsen  recommends  the  following  technique  for  the  study  of  Klossia 
Jielicina  : — 

Break  the  shell  of  an  Helix  Jiortensis  as  far  as  the  second  spiral  near  the 
orifice.  On  removing  the  shell  debris,  a  part  of  the  lung  and  the  pericardium, 
through  which  the  heart  can  be  seen  beating,  will  be  exposed  :  and  lying 
by  the  side  of  these  structures  the  kidney,  appearing  as  a  fusiform  greyish 
mass,  will  be  seen.  Take  hold  of  the  kidney  with  the  forceps,  cut  off  a  small 
piece  with  scissors,  lay  it  on  a  slide  and  cover  with  a  cover-glass. 


766 


PARASITES  IN  TUMOURS 


On  examining  the  preparation  under  the  microscope,  in  addition  to  normal 
epithelial  cells  a  number  of  cells  distended  with  Klossia  will  be  seen  (fig.  369). 
The  parasite  passes  through  all  the  stages  of  its  life  history  in 
the  same  cell :  the  nucleus  of  the  infected  cell  is  often  consider- 
ably hypertrophied  (up  to  60  times  its  natural  size),  and  later 
—when  the  coccidium  has  attained  large  dimensions — the 
nucleus  degenerates  and  atrophies. 

Within  the  oocyst  several  sporocysts  are  formed  :  each  sporo- 
cyst  gives  rise  to  four  sporozoites  and  a  residuum.  When  the 
sporozoi'tes  are  set  free  they  infect  other  epithelial  cells  where 
they  form  schizonts,  which  pass  through  the  ordinary  processes 
of  schizogony. 

The  following  genera,  which  include  no  human  parasite  among 
their  numerous  species,  may  be  mentioned  :  Cyclospora,  Barrouxia, 
Adelea,  Legerella. 


FIG.  369.  — 
Klossia  helicina. 
Kidney  cell  of 
Helix  hortensis 
containing  three 


SECTION  III.— PARASITES  IN  TUMOURS. 
1.  Coccidia. 

The  discovery  a  few  years  ago  of  structures,  which  were  said  to 
bear  a  striking  resemblance  to  the  coccidia,  in  certain  epithelial 
is  situated  near  neoplasms  seemed  to  afford  a  scientific  basis  for  the  parasitic  theory 
the  narrow  end  of  malignant  new  growths :  and  within  a  short  space  of  time  dis- 
(AfterBafbianLJ  coveries  of  protozoa  in  epithelial  tumours  were  reported  from  all 
quarters. 

The  accounts  of  the  numerous  apparently  successful  investigations  which  were 
at  once  set  on  foot  on  receipt  of  these  reports  only  serve  to  show  the  errors  into 
which  one  may  fall  in  interpreting  observed  appearances :  the  result  is  that  these 
researches  are  discredited  and  that  the  majority  of  histologists  have  now  discarded 
the  theory  of  the  coccidial  origin  of  new  growths. 

Many  observers  have  succeeded  in  inoculating  malignant  new  growths  from  one 
animal  into  another  of  the  same  species  and  even  into  animals  of  different  species, 
these  experiments  being  especially  successful  when  a  mouse  cancer  is  inoculated 
into  other  mice.  But  these  inoculations  result  not  in  a  true  infection  but  merely 
in  a  graft  of  cancer  cells,  and  the  growth  which  follows  a  successful  inoculation  is 
in  Jensen's  words  "  a  true  culture  of  cancer  cells."  The  more  important  of  the 
appearances  which  have  been  described  as  parasites  of  new  growths  will  be  here 
briefly  passed  in  review  (vide  also  pp.  707,  735,  839). 

I.  Neisser,  in  1888,  affirmed  the  parasitic  nature  of  Molluscum  contagiosum  or 
Acne  varioliforme :  and  described  certain  peculiar  oviform  structures  which  he 
regarded  as  Coccidia.  These  structures  however  are  merely  cells  undergoing  hyaline 
degeneration. 

In  sections  stained  with  Ranvier's  picro- carmine  it  can  be  seen  that  the  changes 
in  the  cells  increase  progressively  from  the  centre  to  the  surface  of  the  growth.  The 
nuclei  distinctly  visible  in  the  deeper  layers  are  less  conspicuous  in  the  parts  nearer 
the  surface  :  the  section  which  is  stained  yellowish  pink  in  the  centre  becomes  more 
and  more  yellow  as  the  periphery  is  approached  :  the  cells  themselves  are  more  and 
more  infiltrated  with  an  hyaline  substance  which  finally  occupies  the  whole  of  the 
cell  body  including  the  nucleus  :  towards  the  centre  the  hyaline  oval  cells  are  packed 
closely  one  against  another  and  are  surrounded  by  a  filamentous  network  containing 
granules  of  eleidine.  No  indication  of  the  structures  described  and  no  oviform 
parasitic  bodies  can  however  be  made  out,  and  the  only  changes  visible  are  the 
changes  in  the  cells  undergoing  keratinization. 

EL.  Darier,  Malassez,  and  Wickham  have  described  certain  appearances  in  psoro- 
spermosis  follicularis  and  in  Pagefs  disease  of  the  nipple  which,  being  always  situated 
within  the  neoplastic  cells,  they  took  to  be  encysted  coccidia. 

The  structures  described  by  these  authors  are  not  parasites,  but  cells  of  the 
epidermis  derived  from  the  Malpighian  layer  which  after  undergoing  certain  changes 
analogous  to  that  of  normal  keratinization  and  becoming  rolled  up  and  cut  off  from 


PARASITES  IN  TUMOURS 


767 


surrounding  cells  form  cell-nests  (Borrel,  Fabre-Domeyne,  Brault,  Torok  and  others). 
The  micro-chemical  reactions  of  these  bodies  are  those  of  keratinized  cells :  picro- 
carmine  stains  them  bright  yellow,  osmic  acid  deep  brown,  etc. 

III.  Albarran  described  the  occurrence  of  Coccidia  in  a  new  growth  of  the  upper 
jaw.     Albarran' s    "coccidia"    were   in    some  cases  encysted,  and  in  others  not 
encysted  ;    they  were  slightly  rounded,  having  a  more  or  less  visible  nucleus  and 
were  always  situated  outside  the  epithelial  cells. 

These  forms  never  present  the  characteristic  appearance  of  Coccidia  and  never 
show  falciform  bodies.  "  The  different  appearances  presented  by  these  cells,  the 
accumulation  of  refractile  bodies,  the  uniform  staining  of  the  whole  mass  by  the 
same  stain  indicate,  on  the  contrary,  that  they  are  cells  undergoing  disintegration 
preparatory  to  disappearing  "  (A.  Brault). 

IV.  Many  observers  have  described  Coccidia  in  cancers.     The  various  descriptions 
given  by  different  authors  do  not  agree  among  themselves  and  apply  evidently  to 
very    different    conditions.     Soudakewitch,    Foa,    Buffer,    Walker,    Thoma,    and 
Savtchenko  all  ascribe  the  parasitic  forms  which  they  describe  to  the  Coccidia. 
Savtchenko  has,  however,  abandoned  the  idea  that  the  appearances  seen  by  him 
were  due  to  an  animal  parasite  and  considers  rather  that  they  belong  to  the  yeasts. 

Soudakewitch' s  method. — In  110  cancers,  Soudakewitch  found  certain  appearances 
which  he  attributed  to  the  presence  of  Coccidia.  The  technique  employed  in  these 
researches  was  as  follows  : 

1.  Fixation. — Fix  in  a  saturated  aqueous  solution  of  perchloride  of  mercury, 
Flemming's  solution,  or  by  immersion  in  a  1  per  cent,  solution  of  osmic  acid  for 
48  hours  followed  by  3-5  days  in  Miiller's  fluid. 

2.  Embed  in  celloidin. 

3.  Staining,    (a)  Of  sections  fixed  in  perchloride. — Leave  the  sections  in  an  aqueous 
solution  of  safranin  for  1  or  2  days,  then  wash  in  alcohol  slightly  acidified  with 
nitric  acid,  or  in  a  weak  aqueous  solution  of  picric  acid. 

(b)  Of  sections  fixed  in  osmic  acid. — Stain  with  an  old  solution  of  Ranvier's  hsema- 
toxylin. 

4.  Microscopical  appearance. — Use  an  high  power  dry  lens.     In  the  cancer  cells, 
small  rounded  spherical  bodies  will  be  seen  displacing  and  compressing  the  nucleus  : 
these  bodies  have  an  enveloping  membrane,  a  finely  granular  protoplasm  and  a 


FIG.  370.— Parasites  of  cancer.    (After  Soudakewitch.) 


nucleus.  In  size  they  may  be  as  large  as  a  leucocyte,  and  generally  speaking  there 
is  only  one  so-called  parasite  in  each  cell,  though  there  may  be  two,  three,  or 
even  five.  After  staining  with  hsematoxylin  very  diverse  and  complicated  structures 
may  be  seen  in  the  interior  of  the  parasite,  of  which  fig.  370  reproduced  from 
Soudakewitch's  drawings  gives  an  idea. 

Cells  containing  these  structures  are  generally  considerably  hypertrophied  and 
just  about  to  undergo  karyokinetic  division :  occasionally  also  necrosis-  of  the 
nucleus  and  destruction  of  the  cell  protoplasm  is  observed.  'According  to  Soudake- 


768  PARASITES  IN  TUMOURS 

witch  the  parasite  penetrates  into  the  interior  of  the  cell,  and  in  growing  displaces 
and  compresses  against  the  cell  wall  first  the  nucleus  and  afterwards  the  protoplasm  : 
the  parasite  is  set  free  by  the  destruction  of  the  infected  cell,  after  which  the  capsule 
of  the  parasite  itself  bursts  and  the  spores  thus  set  free  infect  neighbouring  cells. 
The  most  important  method  of  propagation  is  said  to  be  that  which  takes  place  in 
the  infected  cell :  the  cancer  cell  divides  by  karyokinesis,  giving  rise  to  two  daughter 
cells,  both  of  which  are  infected. 

Ruffer's  method.  Examination  of  fresh  preparations. — With  a  scalpel  remove  a 
little  of  the  cancer  juice,  transfer  it  to  a  slide,  cover  with  a  cover-glass,  and  examine 
with  an  high  power  dry  objective.  Using  an  Abbe  condenser  rounded  spaces  can  be 
seen  within  some  of  the  epithelial  cells  resembling,  at  first  sight,  vacuoles,  but 
surrounded  by  a  membrane  with  a  double  contour  and  containing  a  body  the 
structure  of  which  is  difficult  to  make  out  with  the  condenser.  If  however  the 
condenser  be  removed  it  will  be  seen  that  this  body  consists  of  a  nucleus  surrounded 
by  a  ring  of  homogeneous  protoplasm. 

Stained  preparations  can  be  obtained  by  mixing  a  drop  of  cancer  juice  with  a 
drop  of  the  stain,  covering  with  a  cover-glass  and  luting  with  paraffin.  The  best 
stain  for  the  purpose  is  an  aqueous  solution  of  methylene  blue  containing  a  little 
aqueous  solution  of  methyl  green  and  very  slightly  acidified  with  acetic  acid.  This 
mixture  stains  the  cell  nucleus  green  and  the  cell  protoplasm  very  pale  blue,  while 
the  nucleus  of  the  parasite  is  stained  pink  and  its  protoplasm  pale  blue. 

Sections.  Fixing. — Place  small  pieces  (4-5  mm.)  of  the  tumour  for  12—24  hours  in 
a  saturated  solution  of  perchloride  of  mercury.  Wash  in  running  water.  Harden 
in  different  strengths  of  alcohol.  Mount  in  xylol-paraffin. 

Staining. — Fix  the  section  on  a  slide,  remove  the  paraffin,  pass  through  alcohol, 
water,  Gram's  solution,  alcohol  and  water  successively.  Stain  by  the  Ehrlich- 
Biondi  method  1  which  stains  the  nuclei  of  the  epithelial  cells  green,  the  nucleolus 
intense  red,  and  the  protoplasm  red ;  while  the  nucleus  of  the  parasite  is  stained 
red  and  its  protoplasm  is  practically  unstained. 

The  following  is  a  better  method : 

(a)  Stain  for  1  or  2  minutes  in  a  5  per  cent,  aqueous  solution  of  hsematoxylin. 
Wash  in  water. 

(&)  Wash  the  section  in  a  concentrated  solution  of  copper  sulphate  until  it  becomes 
black. 

(c)  Transfer  to  a  1  per  cent,  solution  of  hydrochloric  acid  until  the  section  is  pale 
yellow. 

(d)  Wash  again  in  the  copper  sulphate  solution  for  a  few  seconds  until  it  assumes 
a  blue  colour.     Wash  in  a  large  quantity  of  water. 

(e)  Stain  with  an  acid  dye,  e.g.  a  concentrated  tincture  of  cochineal. 
Preparations  stained  in  this  way  show  the  cell-nucleus  blue  and  the  protoplasm 

reddish  blue.     The  parasite  is  red. 

At  the  present  time  very  few  pathologists  regard  the  formations  which  have  just 
been  described  as  Coccidia.  Borrel,  Fabre-Domergue,  Duplay  and  Cazin,  A.  Brault, 
Sikorsky,  De  Quervain  have  all  expressed  themselves  as  opposed  to  the  theory  that 
a  coccidium  is  the  cause  of  cancer.  Without  going  into  details  of  the  objections 
which  they  have  raised  against  the  parasitic  origin  of  malignant  disease  it  may  be 
said  that  the  different  descriptions  which  have  been  given  of  the  supposed  parasites 
in  tumours  are  mutually  conflicting  and  in  no  way  recall  the  forms  characteristic 
of  the  development  of  Coccidia ;  and  that  on  the  other  hand  there  is  a  complete 
morphological  similarity  between  the  pseudo-parasites  and  the  various  appearances 
presented  by  cells  undergoing  degeneration.  According  to  A.  Brault  the  figures 
described  as  Coccidia  in  tumours  are  cellular  modifications  of  one  or  other  of  the 
following  types  :  1.  encapsulation  of  certain  cells  :  2.  hyaline  degeneration  :  3.  pro- 
duction of  endogenous  cells :  4.  excessive  budding  of  the  nuclei :  5.  multiple 
degenerations  of  the  nuclei  and  nucleoli. 

1  The  method  is  as  follows  :  Prepare  saturated  aqueous  solutions  of  methyl- violet,  acid- 
fuchsin  (Rubin  S.)  and  aniline-orange.  Leave  for  3  or  4  days.  Then  mix  100  c.c.  of 
the  orange  solution,  50  c.c.  of  methyl-green  and  20  c.c.  of  acid-fuchsin.  Filter.  Dilute 
1  volume  of  the  mixture  with  60-100  volumes  of  water.  Leave  the  preparations  in  the 
diluted  mixture  for  24  hours,  wash  in  strong  alcohol,  absolute  alcohol  and  xylol  and 
mount  in  balsam. 


MICROCOCCUS  NEOFORMANS  769 

2.  Micrococcus  neoformans. 

Doyen  has  found  in  all  malignant  growths  and  in  many  benign  growths  a  coccus 
which  he  has  named  the  Micrococcus  neoformans.  This  organism  appears  to  be  the 
"  habitiial  parasite  "  of  rapidly-growing  tumours  in  man.  Doyen  has  also  found 
the  same  organism  in  a  rat  (renal  carcinoma),  in  a  mouse  and  in  a  bitch  (cancers  of 
the  mammary  gland). 

The  coccus  of  Doyen,  the  specificity  of  which  is  more  than  doubtful,  can  also  be 
found  on  the  healthy  skin  and  in  glandular  lesions  not  of  a  cancerous  nature.  Borrel 
has  isolated  it  several  times  from  cases  of  tuberculous  mammitis. 

Detection  of  the  parasite. — Doyen  sows  small  pieces  of  tumours  in  a  broth  made 
with  cow's  udder  and  prepared  in  the  same  way  as  ordinary  peptone  broth,  using 
500  grams  of  minced  cow's  udder  freed  from  fat  in  place  of  meat. 

From  a  non- ulcerated  tumour  remove  with  the  necessary  aseptic  precautions 
small  pieces  of  tissue  and  sow  them  in  tubes  containing  about  1  c.c.  of  udder  broth  : 
the  culture  grows  best  when  the  tissue  is  not  completely  immersed  in  the  culture 
medium.  The  coccus  grows  in  16-18  hours  at  37°  C.  but  it  is  not  an  uncommon 
experience  to  find  that  no  growth  is  visible  until  about  the  fifth  day. 

Morphology.— Jf.  neoformans  is  a  very  small  coccus  (0'5-2M  in  diameter),  some- 
times arranged  singly,  sometimes  grouped  in  motile  diplococci  (Isaza),  occasionally 
forming  triads,  tetrads  or  chains  of  4  to  9  irregular  elements. 

It  stains  well  with  the  basic  aniline  dyes  and  is  gram-positive  (at  least  in  young 
forms). 

It  is  a  facultative  aerobe.  On  sloped  agar  it  produces  a  moist  whitish  streak 
which  becomes  glairy  and  ropy  after  2  or  3  days.  It  liquefies  gelatin,  liquefaction 
commencing  about  the  fourth  day  at  20°  C.  (In  this  it  is  differentiated  from  the 
pleomorphic  coccus  of  the  skin  which  does  not  liquefy  gelatin.) 

Experimental  inoculation. — Inoculation  of  M.  neoformans  sets  up  in  most  mice 
and  in  white  rats  after  2  or  3  months  lesions  of  a  neoplastic  nature  which  terminate 
fatally  (Gobert).  The  lesions  experimentally  produced  are  indifferently  of  meso- 
dermic  or  epithelial  origin  (lipomata,  sarcomata,  enchondromata,  adenomata, 
papillomata). 

It  is  best  to  inoculate  into  the  peritoneal  cavity  :  the  lesions  produced  affect 
especially  the  lung,  occasionally  the  liver  and  lymphatic  glands.  In  a  bitch,  Doyen 
produced  two  sub -cutaneous  lipomata  in  the  region  of  the  mammary  glands. 

To  obtain  these  results  Doyen  inoculated  an  emulsion  made  with  fragments  of 
tumours  which  had  been  cultivated  in  broth  for  6  or  7  days  and  the  product  from 
scraping  agar  tubes  sown  with  this  broth  (Gobert).  It  was  therefore  not  a  pure 
culture  which  was  inoculated  but  a  mixture  of  a  pure  culture  and  ground-up  cancer 
tissue. 

Borrel  has  noticed  bronchial  proliferations,  similar  to  those  described  by  Doyen, 
in  rats  which  had  died  spontaneously  with  pulmonary  abscess. 

Toxin. — The  inoculation  of  filtered  glycerin- broth  cultures  into  persons  suffering 
from  cancer  is  followed  by  a  reaction  similar  to  that  which  tuberculin  produces  in 
tuberculous  persons  (Doyen). 

Isaza  proposes  that  this  toxin  should  be  used  in  the  treatment  of  cancer. 

Doyen  has  prepared  a  vaccine  with  M.  neofoimans  and  speaks  favourably  of  the 
results  obtained  in  the  treatment  of  human  cancer.  [Sir  Almroth  Wright  endorses 
Doyen's  opinion  as  to  the  use  of  vaccines  and  thinks  they  relieve  pain.] 


3c 


CHAPTER  LVIII. 
THE  INTRA-CORPUSCULAR  H^MATOZOA. 

Section  I. — The  genus  Hsemamoeba,  p.  770. 

1.  The  haematozoon  of  malaria,  p.  770. 

Methods  of  examination,  p.  770. 

Structure  of  the  parasite,  p.  772.     Morphology,  p.  774.     Life  history,  p.  776. 

The  different  species  of  hsematozoa  found  in  malaria,  p.  778. 

Examination  of  mosquitos,  p.  780. 

Experimental  inoculation,  p.  781. 

2.  The  hsematozoon  of  monkeys,  p.  781.     3.  The  hsematozoon  of  bats,  p.  781. 
4.  The  haematozoa  of  birds,  p.  781. 

Section  II. — The  genus  Hsemogregarina,  p.  783. 

1.  Hcemogregarina    stepanowi,    p.     783.     2.  Hcemogregarina     ranarum,    p.    784. 
3.  Hcemogregarina  lacertarum,  p.  785. 

LAVERAN  classified  the  intra-corpuscular  Hcematozoa  or  Hcemacytozoa  into 
three  genera  :   Hcemamceba,  Piroplasma  and  Hcemogregarina. 


SECTION  I.— THE  GENUS  H^MAMGEBA. 

The  genus  Hcemamceba  or  Plasmodium  consists  of  intra-corpuscular  parasites 
which  are  generally  pigmented  and  in  which  reproduction  takes  place  both 
asexually  (endogenously)  and  sexually  (exogenously),  in  the  latter  case  with 
the  formation  of  flagellated  parasites  representing  the  male  element. 

The  Hcemamcebce  occur  as  parasites  in  man,  monkeys,  bats,  birds  and  some 
reptiles. 

The  various  parasites  described  under  the  names  Hcemamceba,  Plasmodium, 
Laverania,  Proteosoma  and  Halteridium  (Laveran  and  Mesnil)  must  all  be  regarded 
as  belonging  to  the  genus  Hcemamceba. 

1.  The  hsematozoon  of  malaria. 

Hsemamoeba  malarise. 
Synonyms. — (Plasmodium  malar  ice — Plasmodium  vivax — Plasmodium 

prcecox — Laverania  malarice.) 
The  pathogenic  agent  of  malaria  was  discovered  by  Laveran. 

Methods  of  examination. 

The  hsematozoon  should  be  looked  for  in  the  blood  of  infected  persons 
preferably  just  before  or  at  the  commencement  of  the  onset  of  an  attack  of 
fever. 


EXAMINATION   OF  THE  BLOOD  771 

In  certain  cachectic  conditions  the  parasites  are  also  found  in  the  blood  in  the 
intervals  between  the  attacks  of  fever,  but  as  a  rule  they  disappear  from  the  peri- 
pheral blood  during  apyrexial  intervals  especially  when  the  patient  is  being  treated 
with  quinine.  The  crescent  form  of  the  parasite  is  more  resistant  to  this  drug 
than  the  other  forms. 

It  is  best  to  examine  the  blood  in  the  fresh  state. 

(a)  Examination  of  fresh  blood. — The  patient's  finger  is  well  washed  with 
soap  and  water  and  then  with  alcohol  to  remove  the  fatty  secretions  from  the 
skin.     The  pad  of  the  finger  is  then  pricked  with  a  sterilized  needle  and 
after  wiping  away  the  first  drop  of  blood  with  a  piece  of  soft  linen  a  small 
drop  is  collected  on  each  of  a  series  of  scrupulously  clean  glass  slides  and 
immediately  covered  with  a  cover-glass. 

Should  the  drop  of  blood  be  rather  large,  gentle  pressure  may  be  applied 
to  the  cover-glass  and  the  blood  so  squeezed  from  beneath  it  wiped  away  : 
in  this  manner  a  very  thin  layer  of  blood  is  obtained  in  which  the  red  cells 
are  lying  flat  and  not  piled  one  upon  another  in  rouleaux.  In  the  majority 
of  cases  no  advantage  is  gained  by  ringing  the  cover-glass  with  paraffin, 
because  the  blood  will  coagulate  at  the  edges  and  afford  sufficient  protection 
against  evaporation  :  but  in  studying  the  movements  of  the  parasites  it  is 
well  to  adopt  this  precaution  so  that  the  movement  imparted  to  the  red  cells 
by  the  evaporation  taking  place  at  the  edge  is  avoided.  The  preparation 
ought  to  be  examined  with  an  high  power  dry  objective. 

The  living  parasite  may  be  stained  in  the  following  manner.  A  drop  of  methylene 
blue  in  0'75  per  cent,  normal  saline  solution  is  placed  on  the  slide  by  the  side  of 
the  drop  of  blood.  A  cover-glass  is  lowered  on  to  the  two  drops  which  mix  with 
the  result  that  the  parasites  soon  take  up  the  dye — which  is  not  toxic  to  them — 
and  then  stand  out  more  sharply  against  the  unstained  background  (Neveu-Lemaire). 
Celli  and  Guarnieri  use  a  solution  of  methylene  blue  in  ascitic  fluid  for  the  purpose. 

(/3)  Examination  of  dried  films. — Blood  dried  on  cover-glasses  may  also 
be  used  for  investigating  the  presence  of  hsematozoa  and  studying  their 
characters.  The  films  may  be  prepared  on  cover-glasses  in  the  ordinary  way, 
though  sometimes  it  is  preferable  to  spread  the  blood  on  slides  in  the  following 
manner  : — A  little  drop  of  blood  is  placed  at  one  end  of  a  slide  and  spread 
with  the  edge  of  another  slide — a  visiting  card  or  cigarette  paper  will  do 
as  well — by  drawing  the  latter  rapidly  towards  the  other  end  of  t'he  slide 
with  a  single  uninterrupted  motion  (p.  204).  The  preparation  is  dried  as 
rapidly  as  possible  in  the  air  and  then  fixed  in  alcohol-ether  or,  better,  in 
absolute  alcohol  for  10-20  minutes.  Fixation  by  heat  is  too  crude  and 
altogether  unsuitable. 

To  examine  dried  preparations  unstained  the  films  on  the  cover-glasses  are 
simply  laid  on  a  slide  and  fixed  by  their  edges  with  paraffin. 

Methods  of  staining. — The  Hsematozoa  may  be  stained  with  various  dyes, 
methylene-blue,  gentian-violet,  violet-dahlia  and  Boehmer's  haematoxylin 
being  especially  useful. 

Blood  films  prepared  as  above  on  slides  or  cover-glasses  may  be  stained  by 
one  of  the  following  methods. 

1.  Methylene  Blue. — (a)  The  film  is  stained  for  30  seconds  in  a  saturated 
aqueous  solution  of  methylene  blue,  washed,  dried  and  mounted  in  balsam. 
The  red  cells  are  unstained,  the  nuclei  of  the  leucocytes  appear  deep  blue  and 
the  parasites  pale  blue. 

Hanson  recommends  the  use  of  Loaffler's  alkaline  blue  (p.  139).  Koch  prefers 
borax  blue  (borax  5  grams,  distilled  water  100  c.c.  methylene  blue  2  grams).  Stain 
for  30  seconds,  wash  in  water,  dry  and  mount  in  balsam. 

(b)  Stain  in  0'5  per  cent,  solution  of  eosin,  then  in  an  aqueous  solution  of 


772  THE   ILEMATOZOON   OF   MALARIA 

blue  in  the  manner  just  described.  The  preparation  is  thus  double  stained, 
the  red  cells  being  pink,  the  nuclei  of  the  leucocytes  and  the  parasites  blue. 
This  method  is  recommended  by  Laveran. 

(c)  The  films  may  be  stained  with  the  eosin-methylene-blue  mixture  of 
Chenzinsky  (p.  210). 

2.  Violet  stains. — The  films  may  be  stained,  for  a  few  seconds  only,  in  a 
saturated  aqueous  solution  of  gentian-violet  or  violet-dahlia  or  in  Boehmer's 
hsematoxylin.  If  carbol-thionin  be  used  the  staining  should  be  prolonged 
to  5  minutes.  After  staining  the  films  are  washed,  dried  and  mounted  in 
balsam.  The  red  cells  are  unstained  while  the  nuclei  of  the  white  cells  and 
the  parasites  are  stained  violet ;  the  pigment  granules  are  barely  visible. 

Ross's  method  for  the  detection  of  the  parasites. — The  parasites  are  present 
in  the  blood  in  small  numbers  only  as  a  rule,  and  their  detection  in  thin  films 
on  cover-glasses  is  often  a  tedious  undertaking.  Ross  therefore  takes  about 
20  cm.  of  blood  and  spreads  it  in  a  thick  layer  on  a  slide  which,  after  drying 
over  a  flame  but  without  fixing  the  film,  is  washed  in  water  ;  in  this  way  the 
haemoglobin  is  dissolved  and  removed.  The  film  is  now  stained  for  1  minute 
in  a  1  per  cent,  aqueous  solution  of  eosin  followed  by  an  alkaline  solution  of 
methylene  blue  for  15-30  seconds  (1  per  cent,  aqueous  solution  of  methylene 
blue  to  which  0'5  per  cent,  of  carbonate  of  soda  has  been  added  and  heated 
until  it  acquires  a  purple  tint)  :  it  is  finally  washed  in  water,  dried  and  mounted 
in  balsam.  This  method  yields  a  transparent  preparation  in  which  only  the 
leucocytes  and  parasites  are  stained  and  so  allows  of  the  latter  being  readily 
found.  Ruge  advises  the  use  of  Ross's  method,  taking  care  to  fix  the  prepara- 
tion (so  as  to  obviate  the  chance  of  the  blood  being  detached  during  washing) 
in  a  2  per  cent,  solution  of  formalin  containing  1  per  cent,  acetic  acid.  This 
method  of  fixing  in  no  way  interferes  with  the  solution  of  the  haemoglobin. 

Le  Dantec's  method. — In  cases  in  which  the  parasites  are  present  in  very 
small  numbers,  Le  Dantec  advises  hsemolyzing  1  c.c.  of  blood  by  collecting 
it  in  20  c.c.  of  water,  centrifuging  the  mixture  and  examining  the  deposit 
for  parasites. 

Structure  of  the  parasite. 

The  staining  of  the  nuclei  of  Heematozoa  is  difficult  and  requires  special 
methods. 

Laveran's  method.  Recommended. — This  method  is  based  on  the  use  of 
Borrel's  blue. 

Preparation  of  Borrel's  blue. — Dissolve  some  crystals  of  silver  nitrate  in  50  c.c.  of 
distilled  water  in  a  flask  of  about  150  c.c.  capacity.  Fill  the  flask  with  a  10  per  cent, 
solution  of  soda  and  shake.  The  black  precipitate  of  silver  oxide  which  is  thus  obtained 
is  then  carefully  washed  several  times  in  distilled  water  and  after  decanting  the  last 
washing  a  saturated  aqueous  solution  of  medicinal  methylene  blue  (Hochst)  is  added. 
The  solution  is  shaken  several  times  and  allowed  to  stand  for  10  to  15  days ;  the  super- 
natant fluid  which  is  then  decanted  constitutes  Barrel's  blue. 

The  dried  blood  films  after  fixing  for  10  minutes  in  absolute  alcohol  are 
stained  with  the  following  solution,  which  must  be  made  up  just  before  use  : 
Borrel's  blue,1      -  1  c.c. 

O'l  per  cent,  aqueous  solution  of  eosin,2  5     ,, 

Distilled  water,    -          -          -  4     ,, 

Mix  carefully.     The  eosin  and  methylene  blue  solutions  must  be  filtered  just  before  mixing 
but  the  mixture  itself  should  not  be  filtered. 

The  stain  is  poured  into  a  flat  vessel  (Petri  dish  or  special  rectangular  dish) 
and  the  slide  immersed  film  side  downwards,  being  prevented  from  touching 

1  The  solution  of  Borrel's  blue  ought  to  be  prepared  afresh  should  it  rapidly  give  a 
heavy  precipitate  after  mixing  with  the  eosin  solution. 

2  Water-soluble  eosin  (Hochst). 


STRUCTURE   OF  THE   PARASITE  773 

the  bottom  by  means  of  a  little  piece  of  glass  rod  or  a  protuberance  of  the 
vessel ;  otherwise  a  precipitate  is  generally  deposited  on  it. 

If  the  film  has  been  made  on  a  cover-glass  this  may  be  floated,  film  down- 
wards, on  the  surface  of  the  stain  in  a  watch-glass. 

After  staining  for  the  necessary  length  of  time  (5-10  minutes  in  the  case 
of  recently  prepared  films  of  malarial  blood,  and  somewhat  longer  for  older 
films,  and  several  hours  in  the  case  of  the  Hsematozoa  of  birds)  the  films  are 
washed  in  a  liberal  quantity  of  water,  treated  with  a  5  per  cent,  watery  solu- 
tion of  tannin  for  about  2  minutes  and  finally  washed  again  in  distilled  water 
and  dried. 

If  the  staining  be  too  intense  or  if  a  copious  precipitate  be  formed  the 
preparations  mast  be  washed  in  absolute  alcohol  or  clove  oil  and  then  in 
xylol. 

If  the  preparation  is  to  be  kept  it  is  better  not  to  mount  it  in  balsam  or 
cedar  wood  oil,  which  will  soon  dissolve  the  stain. 

The  red  cells  are  stained  pink  and  the  nuclei  of  the  leucocytes  deep  violet  : 
the  protoplasm  of  the  parasite  assumes  a  pale  blue  colour  and  the  chromatin 
of  its  nucleus  becomes  violet  or  purplish  red  :  pigment  granules  (Schiiffner's 
dots)  appear  in  the  bodies  of  the  red  cells  containing  parasites. 

Romanowsky's  method  (p.  210)  is  applicable  to  the  staining  of  Hsematozoa. 
The  nuclei  of  the  parasites  are  stained  purplish-red  and  their  protoplasm  blue. 

This  method  is  merely  of  historical  interest  and  is  now  no  longer  used  in  practice  :  the 
results  are  often  disappointing. 

The  success  of  the  method  depends  upon  the  formation,  under  certain  conditions,  of 
violet  and  azur  from  methylene  blue  ;  these  colour-changes  are  produced  either  by  treating 
methylene  blue  with  a  dilute  alkali  (polychrome  blue)  or  by  obtaining  azur  from  methylene 
blue  by  more  or  less  complicated  processes.  Most  of  the  Romanowsky  methods  have 
been  described  in  connexion  with  the  spirochsete  of  Syphilis  (p.  727)  :  in  this  chapter 
only  those  which  are  especially  applicable  to  the  study  of  the  Hsematozoa  will  be  con- 
sidered. 

Leishman's  stain. — Leishman's  method  is  a  modification  of  that  described 
by  Nocht,  which  depends  upon  the  action  of  sodium  carbonate  on  methylene 
blue. 

Prepare  a  1  per  cent,  aqueous  solution  of  medicinal  methylene  blue  (Grubler)  and 
add  0*5  per  cent,  sodium  carbonate  :  heat  for  12  hours  at  65°  C.  and  keep  for  10 
days  at  the  temperature  of  the  laboratory.  Prepare  also  a  O'l  per  cent,  aqueous 
solution  of  eosin  (eosin  BA  Grubler).  Mix  equal  volumes  of  the  two  solutions  and 
leave  for  10  hours  or  so,  shaking  the  mixture  at  .frequent  intervals.  Filter,  wash 
the  precipitate  with  distilled  water  until  the  washings  are  a  very  pale  blue,  collect 
the  precipitate  on  the  filter,  dry  and  powder.  (Leishman's  stain  in  powder  can  now 
be  obtained  from  Grubler). 

The  staining  solution  is  prepared  by  dissolving  the  powder  in  absolute  methyl 
alcohol  (0*15  per  cent.).  For  use,  3  or  4  drops  of  the  stain  are  poured  on  the  film, 
and  after  30  seconds  6-8  drops  of  water  are  added  and  the  diluted  stain  allowed 
to  act  for  a  further  5-10  minutes.  Wash  in  water  and  leave  the  water  on  the  film 
for  a  minute.  Dry  and  mount  in  balsam.  When  stained  by  this  method  the  red 
cells  are  pale  pink  or  greenish  pink  in  colour,  the  nuclei  of  the  leucocytes  red,  the 
Hsematozoa  blue  and  their  chromatin  ruby-red. 

Tenner's  stain. — This  method,  like  that  of  Wright,  is  based  upon  the  use 
of  the  product  obtained  by  the  interaction  of  solutions  of  methylene  blue 
and  eosin.  The  stain  is  sold  in  the  dry  form  by  Grubler. 

The  film,  which  does  not  require  fixing,  is  stained  for  3-5  minutes  in  the  following 
solution. 

Jenner's  stain  in  powder  (Grubler),          ....  l  gram. 

Pure  absolute  methyl  alcohol,         -  -         100  c.c. 

The  preparation  should  be  covered  during  the  staining  process  to  prevent  evaporation 
of  the  alcohol. 


774  THE   ELEMATOZOON   OF  MALARIA 

Wash  rapidly  (for  a  few  seconds)  in  distilled  water.  Dry.  The  preparation  may 
be  examined  and  preserved  at  this  stage  or  may  be  mounted  in  balsam.  The  colour 
reactions  are  the  same  as  with  Irishman's  stain. 

Giemsa  's  stain.  —  This  method  is  based  upon  the  use  of  a  mixture  of  solutions 

of  azur  II  and  eosin,  and  has  been  described  at  p.  727.     Laveran  modified 

the  technique  somewhat  to  make  it  applicable  to  the  staining  of  Hsematozoa. 

The  dried  blood  film  is  fixed  in  absolute  alcohol  and  then  stained  for  10  minutes  in 

the  following  solution  : 

0-1  per  cent,  aqueous  solution  of  eosin,  -         -  2  c.c. 

Distilled  water,    -  8     „ 

0-1  per  cent,  aqueous  solution  of  azur  II,         -  1     „ 

Wash  in  water,  treat  for  2  minutes  with  a  5  per  cent,  aqueous  solution  of  tannin. 
Wash,  dry  and  mount. 

Marino's  stain.  —  This  method,  which  has  already  been  described  at  p.  727, 
may  be  used  to  stain  the  Hsematozoa.  It  is  based  upon  the  use  of  a  mixture 
of  eosin,  methylene  blue  and  azur. 

Morphology. 
Appearance  in  human  blood. 

In  the  blood  of  persons  infected  with  malaria  the  hsematozoon  assumes  one. 
of  the  following  forms. 

1.  The  mtra-corpuscular  amoeboid  form  (Spherical  body)  [Ring  parasite].  — 
The  amoeboid  trophozoites  within  the  red  cells  are  the  forms  most  commonly 
seen.  They  are  small  spherical  structures  1—  G/A  in  diameter,  composed  of  an 
hyaline,  colourless,  transparent  protoplasm,  and  since  they  exhibit  amoeboid 
movements  are  sometimes  known  as  the  amoeboid  bodies.  As  a  rule  they 
appear  like  small  clear  specks  attached  to  the  red  cells.1  Two,  three  and 
even  four  parasites  may  be  present  in  one  red  cell.  Occasionally  they  are 
found  free  in  the  serum. 

Even  the  smallest  of  the  amoeboid  bodies  sometimes  contain  one  or  two 

grains  of  black  pigment  (melanin),  and  as  the 
parasite  increases  in  size  pigment  becomes 
more  abundant.  The  pigment  grains  are 
sometimes  arranged  like  a  wreath  around 
the  margin  of  the  amoeboid  body  and  at 
other  times  dotted  irregularly  through  the 
substance  :  they  often  show  active  move- 
ment which  is  more  irregular  and  less 
constant  than  Brownian  movement. 

The  amoeboid  bodies  have  in  addition  a 
nucleus,  situated  excentrically,  attached  to 
the  periphery,  and  difficult  to  stain.  In 

preparations  stained  with  methylene  blue, 
FIG.  371.  —  The  malarial   parasite   In      S.  i  r  J.T.  -±.     •          i 

human  blood,     x  looo.    a,  The  young     tne  protoplasm  of  the  parasite  is  coloured 


SS^SPSTuS  am<Eboid'  or  ™«  Para-     blue  while  the  nucleus  is  represented  by  a 
site;  b,  at  a  later  stage;  c  and  d,  stages        ,  ,          ^  .    .       ,   ,       £  ,J  , 

in  the  segmentation  of  the  schizont;  e,     clear  vacuole.     Stained  by  Komanowsky  s 

SZlDLg?ammaticT  segmenting  schi'     or  Laveran's  method  a  chromatin  granule 

within  the  nucleus  is  stained  violet. 

In  fresh  blood  films,  at  the  end  of  half  to  three-quarters  of  an  hour  the 
amo3boid  movements  cease  and  the  parasite  dies  ;  the  margins  then  become 
irregular  and  the  grains  of  melanin  accumulate  irregularly  in  various  parts 
of  the  parasite. 

1  [This  is  now  considered  to  be  merely  an  appearance.  The  parasites  are  believed  to- 
be  always  within  the  red  cells.  ] 


MORPHOLOGY  775 

2.  The  rosette  or  marguerite  form. — The  rosette,  marguerite  or  segmenting 
forms  represent  the  schizogonous  mode  of  reproduction  of  the  Hsematozoon 
of  malaria.  They  are  only  met  with  in  very  small  numbers  in  chronic  con- 
ditions and  are  best  looked  for  during  the  early  stages  of  an  attack  of  fever  : 
sometimes  they  are  not  to  be  found  in  the  peripheral  blood  but  only  in  the 
liver  and  spleen. 

In  some  of  the  amoeboid  bodies  the  margins  will  be  seen  to  be  slightly 
indented  and  the  pigment  grains  collected  in  the  centre  of  the  parasite — this 
is  the  first  stage  of  schizogony.  The  periphery  soon  becomes  more  deeply 
indented  and  gives  the  parasite  a  marguerite  appearance  :  each  segment  then 
separates  from  its  neighbours,  in  such  a  way  that  a  number  of  little  spherules 
(merozdites]  are  formed,  none  of  which  contain  pigment — pigment  being  only 
found  in  the  adult  forms  of  the  parasite. 

The  number  of  segments  into  which  the  marguerite  form  breaks  up  is  very 
variable  (six  to  twenty).  According  to  Golgi,  those  breaking  up  into  eight 
segments  are  found  in  quartan  fever,  those  giving  rise  to  sixteen  or  twenty 
spherules  in  tertian  fever  (p.  180). 


I, 


FIG.   372.  —  The  hsematozoon   of    malaria.  FIG.  373. — The  haematozoon  of  malaria, 

x  1000.    a,  the  crescent  form  of  macrogamete ;  x  1000.    a,  macrogamete ;  b,  microgametes 

b,  the  crescent  form  of  microgametocyte  (Plas-  separating  from  the  microgametocyte  (fla- 

modium  -prcecox).     (Diagrammatic.)  gellated  body).    (Diagrammatic.) 

3.  The  crescent  form. — Crescents  are  seen  in  the  blood  of  persons  who  have 
been  infected  for  a  long  time  and  are  suffering  from  malarial  cachexia. 

The  crescent-shaped  parasites  measure  8-9/x  long  by  about  2//,  broad  : 
their  protoplasm  is  transparent  and  colourless  except  for  a  collection  of  black 
pigment  grains  in  the  centre.  On  their  concave  side  a  very  fine  line  is  often 
seen  connecting  the  two  horns  of  the  crescent.  Crescents  are  either  found 
free  in  the  serum  or  attached  to  red  cells. 

Laveran  was  the  first  to  show  that  these  crescents  later  change  their  shape, 
becoming  first  oval  then  spherical.  Crescents  represent  a  stage  in  the  sexual 
life-history  (sporogony)  of  the  Hsematozoon  (vide  post). 

4.  Flagellated  bodies. — At  the  periphery  of  certain  medium-sized  spherical 
parasites  motile  filaments  (flageUa)  are  sometimes  seen  which  are  endowed 
with  considerable  powers  of  movement  and  displace  the  red  cells  in  their 
neighbourhood.     The  flagella  are  three  or  four  times  the  diameter  of  a  red 
cell,  but  so  transparent  and  delicate  that  when  at  rest  it  is  almost  impossible 
to  see  them.     One  to  four  flagella  may  originate  from  a  single  spherical  body 
and  these  may  be  arranged  symmetrically  around  the  parasite  or  grouped 
together  at  the  same  point.     The  flagella  move  independently  of  each  other, 
and  their  displacement  often  imparts  some  slight  movement  to  the  spherical 


776  THE   ELEMATOZOON   OF  MALARIA 

body  ;  sometimes  it  is  a  mere  movement  of  oscillation,  sometimes  a  true 
movement  of  translation. 

Certain  flagella  show  a  slight  pear-shaped  swelling  at  their  free  extremities. 

At  a  given  moment  the  flagella  detach  themselves  from  the  spherical  bodies 
and,  becoming  free,  move  about  among  the  red  cells  in  the  field  of  the  micro- 
scope. As  soon  as  the  flagella  have  separated  the  spherical  bodies  lose  their 
shape  and  the  grains  of  pigment  in  them  collect  together  in  a  mass. 

Morphologically,  the  flagellated  bodies  represent  microgametocytes  and  the 
flagella  the  microgametes  (vide  infra).  They  are  never  formed  in  the  circulating 
blood,  but  appear  very  soon  in  blood  which  has  been  withdrawn  from  the 
vessels  and  equally  quickly  disappear  from  it.  Their  complete  life-history 
can  only  take  place  in  the  alimentary  canal  of  certain  mosquitos. 

The  method  of  examining  blood  for  flagella. — In  examining  blood  for  the  flagellated 
forms  of  the  parasite  it  is  advisable  to  let  the  blood  stand  for  a  few  minutes  after  it 
leaves  the  body.  Collect  a  small  drop  of  blood  on  a  slide  which  has  been  moistened 
by  breathing  upon  it,  and  if  possible  from  a  patient  in  whose  blood  the  crescent 
forms  are  numerous,  spread  it  rapidly  with  a  needle  and  invert  it  on  to  a  moist 
chamber — made  by  placing  a  thick  fold  of  blotting-paper  soaked  in  water,  and 
from  the  centre  of  which  a  rectangular  piece  about  2 '5  cm.  x  1/5  cm.  has  been  cut 
out,  on  a  sheet  of  glass.  Manson  recommends  leaving  the  slide  under  these  con- 
ditions for  15—15  minutes,  then  dry  in  the  flame,  fix  in  absolute  alcohol,  wash 
in  20  per  cent,  solution  of  acetic  acid  in  water  to  dissolve  the  haemoglobin,  wash  in 
water,  stain  according  to  the  directions  given  above,  dry  and  mount. 

The  life-history  of  the  malarial  parasite. 

Like  the  Coccidia,  the  hsematozoon  of  malaria  has  two  methods  of  repro- 
duction. 

1.  Asexual  reproduction  or  schizogony,  which  occurs  in  the  blood  of  the 
human  subject. 

2.  Sexual  reproduction  or  sporogony,  which  takes  place  outside  the  human 
body  in  the  alimentary  canal  of  certain  mosquitos  of  the  genus  Anopheles 
(Ross,  MacCallum,  Laveran,  Manson,  Grassi,  Bignami  and  Bastianelli,  and 
others). 

(a)  Schizogony  or  asexual  cycle.— The  intra-corpuscular  amoeboid  parasite 
— spherical  body  or  schizont — having  reached  maturity  undergoes  schizogony. 
The  nucleus  divides  into  a  number  of  daughter  nuclei  which  pass  towards 
the  periphery  of  the  parasite,  and  the  protoplasm  also  divides  by  means  of 
sulci  passing  in  from  the  periphery ;  thus  the  schizont  assumes  a  daisy  or 
marguerite  appearance,  the  pigment  accumulates  in  the  centre  and  the  seg- 
ments marked  off  by  the  segmentation  of  the  daisy-shaped  body  constitute 
the  merozoites.  These  are  set  free  in  the  blood  stream  by  the  rupture  of  the 
red  cell  and  attaching  themselves  to  other  red  cells,  penetrate  the  latter, 
grow,  become  pigmented  and  again  form  adult  schizonts. 

Schizogony  occurs  over  and  over  again,  and  by  this  process  of  endogenous 
reproduction  the  parasite  multiplies  in  the  tissues  with  extreme  rapidity  : 
in  tertian  fever,  for  instance,  a  new  generation  is  produced  every  other  day. 
The  onset  of  the  fever  coincides  with  the  discharge  of  the  merozoites  into 
the  blood  stream. 

(/2)  Sporogony  or  sexual  cycle.— Besides  the  amoeboid  bodies  and  the 
daisy  forms  (schizonts),  gametes  represented  in  one  species  by  crescents,  and 
in  other  species  by/ree  spherical  bodies  larger  than  the  schizonts,  are  found 
in  the  blood  of  malarial  patients  :  these  gametes,  whether  crescent-shaped 
or  spherical,  are  derived  "  from  merozoites  which,  exhausted  by  a  long  series 
of  schizogonic  multiplications,  have,  for  this  reason,  undergone  evolution  in 
another  direction  "  (Blanchard). 


LIFE   HISTORY  777 

Gametes  cannot  multiply  in  the  blood  of  man  and  can  only  propagate  their 
species  in  the  digestive  tube  of  certain  mosquitos.  In  the  crescent  forms  of 
gametes  two  kinds  can  be  distinguished ;  the  female  gametes  or  macrogametes 
(fig.  374,  E)  which  are  longer,  more  slender  and  have  the  pigment  more  closely 
concentrated  about  the  nucleus,  and  the  male  gametes  or  microgametocytes 
(fig.  374,  D)  which  are  shorter  and  stouter  and  have  the  pigment  more  scattered 
through  the  cell  protoplasm. 

[In  the  spherical  forms  of  gametes  the  male  and  female  cells  are  not  so 
easily  distinguished  though  if  the  male  cell  be  watched  in  a  fresh  preparation 
under  the  microscope  it  will  be  seen  in  due  course  to  extrude  its  flagella.] 


X-1    X. 

I       \ 


*•*       » 


G 


FIG.  374. — The  hsematozoon  of  malaria.  Stained  by  Irishman's  method. — 
Amoeboid  or  ring  parasites :  gametes :  crescents :  flagellated  bodies.  A,  B,  C, 
amoeboid  trophozoi'tes  in  a  red  cell ;  D,  male  crescent ;  E,  female  crescent ; 
F,  male  spherical  gamete  ;  G,  macrogamete  ;  H,  flagellated  body  or  micro- 
gametocyte  ;  I,  fertilization  of  a  macrogamete  by  a  microgamete  ;  J,  zygote. 

When  a  female  mosquito  of  the  genus  Anopheles  (the  males  do  not  bite) 
bites  a  person  suffering  from  malaria  it  withdraws  a  certain  amount  of  blood 
containing  the  various  forms  of  the  parasite  which  have  already  been 
described  :  in  the  alimentary  canal  of  the  mosquito  the  young  amoeboid 
parasites  and  schizonts  are  rapidly  destroyed  but  the  gametes  survive  and 
undergo  sexual  reproduction.  The  macrogametes,  or  female  cells  become 
spherical  and  have  a  small  irregular  centrally  situated  mass  of  chromatin 
(fig.  374,  G).  In  the  microgametocytes,  or  male  cells,  the  chromatin  splits, 
generally  into  four  secondary  nuclei,  (fig.  374,  F)  which  travel  towards  the  peri- 
phery, protrude  from  the  protoplasm  of  the  cell  and  become  drawn  out  into 
long,  slender,  motile  filaments,  flagella  or  microgametes  (fig.  374,  H),  which 
soon  become  detached  and  go  in  search  of  the  macrogametes.  After  the 
flagella  are  detached  the  microgametocyte  soon  dies.  The  microgametes  meet 
the  macrogametes  in  the  stomach  of  the  mosquito,  penetrate  and  fertilize 
them  (fig.  374,  I)  :  the  fertilized  macrogamete  constitutes  the  zygote. 

The  young  zygote,  spherical  in  the  first  instance,  elongates  (fig.  374,  J), 
moves  towards  the  wall  of  the  stomach  of  the  mosquito  and  passes  between  the 


778  THE   ILEMATOZOON  OF  MALARIA 

epithelial  cells  lining  the  mucous  membrane,  again  changes  to  the  spherical 
shape  and  forms  a  small  cyst  in  the  muscular  wall.  Measuring  at  first 
6/A  in  diameter  it  gradually  grows,  lifting  the  outer  wall  of  the  stomach  and 
forming  an  hernia  into  the  body  cavity  of  the  insect,  until  it  ultimately 
attains  a  diameter  of  60-80/z.  Its  chromatin  is  collected  into  a  central  mass. 
Changes  now  begin,  culminating  in  the  formation  of  the  oocyst.  The 
chromatin  splits  into  numerous  small  fragments  which,  passing  to  the  peri- 
phery and  becoming  surrounded  with  protoplasm,  form  the  sporoblasts 
(fig.  375).  From  the  sporoblasts  are  formed  the  sporozo'ites ,  which  are  at  first 
spherical  but  subsequently  elongate  and  become  pointed  at  the  ends.  Then 
the  oocyst  bursts  (fig.  376)  and  the  sporozo'ites  are  set  free  into  the  body  cavity 
of  the  mosquito,  from  whence  they  are  swept  along  by  the  circulation  into  the 
thorax  and  head  and  invade  especially  the  salivary  glands.  When  an 
Anopheles  mosquito  thus  infected  bites  a  healthy  individual  it  inoculates 
into  the  victim's  blood  both  its  venin  and  these  sporozoites.  The  sporozoites 
act  as  merozoi'tes,  infect  the  red  cells  and  give  rise  to  intra-corpuscular 
spherical  amoeboid  parasites. 


FIG.  375. — The  hsematozoon  of  malaria.  FIG.  376. — The  haematozoon  of  malaria.    A 

Ripe  oocyst.    (After  Grassi.)  ruptured  oocyst  from  which  the  sporozo'ites  are 

escaping.     (After  Grassi.) 

The  different  species  of  haematozoa  found  in  malaria. 

Golgi,  Grassi  and  Feletti,  and  other  Italian  observers  have  for  a  long  time 
considered  that  there  is  more  than  one  species  of  the  malarial  hsematozoon 
and  the  majority  describe  three,  corresponding  to  the  parasites  of  quartan, 
tertian  and  the  irregular  tropical  fevers.  Manson  however  considers  that 
there  are  five  species  corresponding  to  as  many  different  clinical  types  of 
the  disease. 

In  the  present  chapter  Golgi's  classification  will  be  followed. 

FIG.  377. — Life  history  of  the  malarial  parasite  (after  Grassi  and  Schaudinn). 

A.  Schizogony.     I.  A  free  sporozoite.     2.  A  sporozoite  entering  a  red  cell. 
3,  4,  5,  6.  Growth  of  the  intracorpuscular  amoeboid  parasite  preceding  multipli- 
cation and  division  into  merozoites.    7.  Merozoites  which  have  burst  the  red  cell 
and  become  free  in  the  blood  stream.     Some  merozoi'tes  penetrate  other  red 
cells  and  pass  through  the  same  series  of  changes  (1-7)  others  become  gametes. 

B.  Sporoaony.—Q&'Usi.  Growth  and  differentiation  of  the  female  cell  or 
macrogamete— 9b-13b.  Growth  and  differentiation  of  the  male  cell  or  micro- 
gametocyte.     14b.  Budding  off  of  microgametes  from  the  microgametocyte. 
15b.  Free  microgamete  or  flagellated  body.— 16.  Fertilization  of  the  macro- 
gamete  by  the  microgamete.    17.  The  fertilized  cell  or  zygote  (ookinete).     18. 
Infection  of  the  epithelial  cells  lining  the  walls  of  the  mosquito's  stomach.     19-24. 
Growth  and  development  of  the  oocyst  and  formation  of  sporozoites  within  it. 
25-27.  Rupture  of  the  oocyst,  and  discharge  of  the  sporozoites  which  travel  to 
the  salivary  glands  of  the  mosquito .     1 .  Discharge  of  a  sporozoite  from  the  mouth 
dunng  biting.    (From  Mense's  Handbuch  der  Tropenkrankheiten.) 


780  THE   ILEMATOZOON   OF  MALARIA 

1.  Plasmodium  malarias. — This  is  the  parasite  of  quartan  fever  :    its  life- 
cycle  occupies  72  hours,  and  the  young  forms  exhibit  very  slow  amoeboid  move- 
ments.    The  intra-corpuscular  amoeboid   bodies  are  smaller  than  a  normal 
erythrocyte.     The  pigment  grains  are  numerous  and  large  and  show  very  little 
movement.     The  parasite  forms  a  rosette  made  up  of  6  to  12  merozoiites  with 
the  pigment  all  collected  in  the  centre :  it  does  not  form  crescent  gametes. 

2.  Plasmodium  vivax. — The  parasite  of  benign  tertian  malaria  ;    its  life- 
cycle  occupies  48  hours,  and  the  young  forms  show  more  active  movements 
than  the  young  forms  of  P.  malarice.     The  amoeboid  forms  are  larger  than  a 
red  blood  corpuscle,  and  the  infected  cell  hypertrophies,  becomes  pale  and 
is  filled  with  characteristic  fine  red  granules  (Schuffner's  dots).     The  parasite 
contains  numerous,  fine,  highly  motile  grains  of  pigment :    the  rosette  is 
formed  of  12  to  20  merozoi'tes  :   no  crescent  gametes  are  formed. 

3.  Plasmodium  preecox  (Laverania  malarice). — The   parasite   of  tropical, 
pernicious,    irregular,    quotidian,    malignant    tertian    and    sestivo-autumnal 
fevers.     Young  forms  show  very  active  amoeboid  movement.     The   intra- 
corpuscular  parasites  are  so  small  that  a  single  red  cell  can  contain  several 
of  them  :  the  pigment  grains  are  scanty,  fine,  and  exhibit  little  motility  :  the 
rosette  is  constituted  of  6  to  15  small  merozo'ites  :   the  gametes  are  crescent- 
shaped.     The  infected  blood  cells  retain  their  natural  size  and  assume  a  deep 
copper  colour.     Parasites  are  only  present  in  small  numbers  in  the  peripheral 
blood  but  abound  in  the  blood  of  the  internal  organs.     The  period  of  the 
life-cycle  is  irregular  and  may  be  24  or  48  hours  or  longer. 

Laveran  holds  that  the  parasites  found  in  malaria  are  not  different  species 
but  merely  varieties  of  the  same  species  and  distinguishes  three  such  varie- 
ties : — Hcemamceba  malarice  var.  parva,  tertiana,  quartana.  Laveran  has 
seen  changes  taking  place  which  he  regards  as  a  change  from  one  variety  to 
another.  Hcemamceba  malarice  parva  corresponds  to  Laverania  malarice,  the 
parasite  of  the  pernicious  and  tropical  fevers.  In  Laveran's  opinion  clinical 
observation  and  microscopical  investigation  agree  in  showing  that  all  forms 
of  malaria  are  clinically  and  setiologically  the  same  disease. 

MetchnikofT  and  van  Gorkone  accept  Laveran's  views.  According  to  van 
Gorkone  the  variations  in  size,  motility  and  morphological  appearance  are 
due  to  the  rapidity  of  growth  of  the  Hsematozoa,  which  depends  upon  whether 
the  blood  of  the  person  infected  is  relatively  favourable  to  their  development 
or  not. 

The  examination  of  mosquitos.1 

Neveu-Lemaire  recommends  the  following  method. 

A  few  drops  of  chloroform  are  poured  on  the  wool  plug  of  the  tube  containing  the 
insect.  When  the  mosquito  is  anaesthetized  the  legs  and  wings  are  pulled  off  and 
the  body  laid  on  a  slide. 

To  withdraw  the  stomach  fix  the  mosquito  by  pressing  with  a  needle  at  the  junction 
of  the  thorax  and  abdomen,  then  with  a  second  needle  pull  gently  on  the  last  two 
segments  of  the  abdomen,  holding  the  needles  horizontally  :  the  stomach  ruptures 
at  its  junction  with  the  oesophagus  and  is  withdrawn  with  the  intestines.  It  may  now 
be  examined  in  one  or  other  of  the  following  solutions  : 

1.  Commercial  formalin, 2  grams. 

Distilled  water,  .          .         iQQ 

2.  Sodium  chloride.  ....  j  -59  grams. 
The  white  of  one  egg. 

Distilled  water,  250 

Filter  before  use. 

1  For  a  detailed  account  of  the  classification  of  mosquitos  and  of  the  methods  of  examina- 
tion the  reader  is  referred  to  Moustiques  et  maladies  infectieuses  by  Ed.  and  £t.  Sergent. 

[A  full  account  of  the  Structure  and  Biology  of  Anopheles  is  given  by  Nuttall  and 
Shipley  in  the  Journal  of  Hygiene,  vol.  i.  (Camb.  Univ.  Press)  ] 


EXAMINATION   OF  MOSQUITOS  781 

The  preparation  should  first  be  examined  under  a  low  power  of  the  microscope 
and  when  a  cyst  is  found  a  higher  power  or  a  ^  immersion  lens  is  turned  on. 
If  there  is  still  some  blood  in  the  stomach  it  may  be  mixed  with  a  0'75  per  cent, 
saline  solution  :  the  blood  flows  into  the  liquid  and  the  preparation  is  covered  with 
a  cover-glass  and  examined  for  parasites. 

The  examination  of  the  salivary  glands  is  more  difficult,  and  the  dissection  should 
be  done  with  a  lens.  Fix  the  middle  of  the  thorax  by  laying  a  needle  horizontally 
across  it.  then  with  a  second  needle  gently  tear  away  the  head,  taking  the  three 
lobes  of  the  salivary  gland  with  it. 

The  glands  should  be  examined  in  the  following  solution : 

Distilled  water,    -          -  -         100       grams. 

Commercial  formalin,  2  ,, 

Sodium  chloride,  0*75  gram. 

Staining  method. — Dissect  the  stomach  in  a  0'75  per  cent,  solution  of  sodium 
chloride  :  fix  for  1  minute  in  osmic  acid  vapour,  stain  with  picro-carmine  and  mount 
in  glycerin. 

Any  parasites  which  may  be  present  in  the  undigested  blood  in  the  stomach  can 
be  examined  by  preparing  a  blood  film  with  a  drop  of  the  saline  solution  in  which 
the  stomach  was  dissected  and  staining  in  the  manner  described  on  p.  771. 

The  sporozoi'tes  can  also  be  examined  by  gently  pressing  upon  and  rupturing  one 
of  the  oocysts  into  the  saline  solution  in  which  the  stomach  was  dissected  and  then- 
examining  a  drop  of  the  fluid  under  the  microscope. 

Sections. — The  best  method  is  to  fix  the  whole  mosquito.  After  removing  the 
legs  and  wings  pour  a  little  boiling  acid  perchloride  (p.  189)  on  to  it.  The  body 
will  break  up  into  two  or  three  pieces  which  can  then  be  embedded  in  paraffin. 
The  sections  must  be  cut  very  thin  and  should  be  stained  with  Bcehmer's  or  Heiden- 
hain's  hsematoxylin. 

Experimental  inoculation. 

The  disease  can  be  reproduced  experimentally  by  inoculating  the  parasite 
into  man  :  to  ensure  the  success  of  the  experiment  it  would  appear  best  to 
inoculate  infected  blood  into  a  vein.  Eight  to  ten  days  after  the  inoculation 
parasites  appear  in  the  blood  and  symptoms  of  malaria  develop. 

Man  can  be  also  experimentally  infected  with  malaria  by  being  bitten  by 
an  Anopheles  which  has  sucked  the  blood  of  infected  persons.  Manson 
infected  his  son  [and  his  laboratory  attendant]  neither  of  whom  had  ever 
been  out  of  England,  by  allowing  them  to  be  bitten  by  mosquitos  fed  on 
malarial  blood  sent  from  Rome. 

Monkeys  are  immune  to  the  human  parasite  and  all  attempts  to  infect  the 
lower  animals  have  failed. 

It  has  not  been  possible  to  cultivate  the  hsematozoon  outside  the  body. 

2.  The  hsematozoon  of  monkeys. 

Hsemamceba  kochi. 

Koch,  Kossel,  Bruce  and  Nabarro,  and  Laveran  have  described  an  intra- 
corpuscular  hsematozoon  in  several  species  of  monkeys  but  chiefly  in 
the  Cercopitheci.  In  the  fully  grown  form  the  parasite  appears  as  a  spherical 
pigmented  body  :  a  rosette  stage  has  not  been  seen.  Monkeys  cannot  be 
experimentally  infected. 

3.  The  hsematozoon  of  bats. 

Dionisi  has  found  an  Hsematozoon  of  the  genus  Hsemamoeba  (H.  melani- 
phera)  in  bats  (Miniopterus  scJireibersii). 

4.  The  hsematozoa  of  birds. 

Hsematozoa  closely  related  to  the  hsematozoa  of  malaria  have  been  found 
in  the  blood  of  many  birds  (jays,  magpies,  rooks,  crows,  hawks,  screech  owls, 


782  THE   HSEMATOZOA   OF   BIRDS 

owls,  pigeons,  chaffinches,  larks,  etc.).  These  parasites,  which  have  been 
variously  described  as  Halteridium,  Hcemamcjeba,  Proteosoma,  Laverania  and 
Plasmodium,  have  been  investigated  by  Danilewsky,  Laveran,  and  by  Grassi 
and  Feletti.  According  to  Laveran  they  should  all  be  included  in  the  genus 
Hcemamosba. 

Though  the  Hsematozoa  found  in  different  birds  resemble  each  other  very  closely, 
Laveran  considers  that  they  represent  several  species  : — 
Hcemamceba  relicta  (PL  relictum,  Proteosoma  of  Labbe) : 
Hcemamceba  danilewskyi  (Halteridium  danilewskyi,  PL  danilewskyi) : 
Hcemamceba  ziemanni  (PL  ziemanni) : 
Hcemamceba  majoris. 

The  Hsematozoa  of  birds  are  generally  attached  to  the  surface  of  or  contained 
within  the  red  cells.  Most  commonly  the  parasites  are  spherical  but  occasionally 
ovoid :  they  alter  the  shape  of  the  infected  blood  cell  which  they  gradually  destroy 
and  thus  become  free  in  the  blood.  In  the  mature  condition  they  may  assume  one 
of  two  forms  and  these  have  been  studied  in  the  case  of  Hcemamceba  danilewskyi  by 
MacCallum,  Opie,  and  by  Marchoux  and  Laveran. 

1.  Finely  granular  forms,  staining  well  with  methylene  blue  and  containing 
scattered  grains  of  pigment.  Stained  by  Laveran's  method  (p.  772)  the  nucleus  is 
seen  to  be  rounded  or  oval,  situated  towards  the  centre  of  the  parasite  and  containing 

a. small  karyosome:  the  nucleus  is  stained 
violet  and  the  karyosome  a  deep  violet. 
These  represent  the  female  elements. 

2.  Hyaline  forms  containing  large  granules 
of  pigment  at  the  extremities.  These  stain 
feebly  with  blue,  and  have  a  large  very 
elongated  nucleus  with  irregular  outline  and 
occupying  the  whole  of  the  centre  of  the 
parasite :  after  leaving  the  red  cell,  these 
parasitic  forms  assume  a  spherical  shape 
and  give  origin  to  .flagella.  They  represent 
the  male  elements.  The  flagella  are  4  to  6 
in  number  on  each  parasite  and  have  an 
enlargement  which  varies  in  shape  and 
position  :  situated  near  the  enlargement  is  a 
mass  of  chromatin. 

After  separating  from  the  microgameto- 
cyte  the  flagella  meet,  penetrate,  and  fertilize 
cytes.  the  female  cells. 

Segmented  bodies  or  rosette  forms  are  seldom 

seen  (Danilewsky)  and  have  never  been  met  with  in  H.  danilewskyi  (Laveran)  but 
are  found  in  H.  relicta. 

H.  relicta  is  a  common  parasite  of  sparrows  in  the  Roman  Campagna.  The 
examination  of  a  drop  of  blood  from  an  infected  bird  shows  adult  forms,  spherical 
or  oval  and  pigmented,  young  unpigmented  forms,  segmented  bodies,  and  micro- 
gametocytes  extruding  flagella.  Ross  has  shown  that  H.  relicta  passes  a  part  of 
its  life-cycle  in  certain  mosquitos  (Culex  pipiens)  and  that  the  infection  is  transmitted 
by  the  bites  of  these  insects. 

It  is  not  proved  that  the  Hsematozoa  found  in  birds  produce  disease  or  cause  fever. 
As  a  rule  birds  infected  with  hsematozoa  do  not  show  any  symptoms  of  disease, 
but  Danilewsky  has  shown  that  at  certain  times  the  birds  become  ill  and  may  even 
die,  and  that  in  these  cases  rosette  forms  are  found  in  the  blood. 

Birds  cannot  be  infected  with  the  human  Hsematozoa,  but  are  susceptible  to 
infection  with  infected  blood  from  birds  of  the  same  species  (Celli  and  Sanfelice, 
Laveran). 

Mattei  failed  to  produce  infection  by  inoculating  infected  pigeon's  blood  into  the 
veins  of  a  man. 

Quinine  though  eminently  efficient  against  the  Hsematozoa  of  man  has  no  action 
on  the  Hsematozoa  of  birds. 

Methods  of  examination. — Blood  is  obtained  by  pricking  one  of  the  veins 


H^EMOGREGARINA  STEPANOWI  783 

in  the  fold  of  the  wing  with  a  needle  after  plucking  a  few  feathers.  The 
blood  should  be  collected  in  a  pipette. 

[Manson  says  :  "  The  pad  of  the  terminal  phalanx  of  the  bird's  toe  is 
cleaned  with  spirit,  dried  and  deeply  pricked  with  a  needle  :  a  droplet  of 
blood  is  then  expressed  and  mounted  in  the  usual  ways."] 

Films  should  be  prepared  and  treated  in  the  same  way  as  human  malarial 
blood  (p.  771). 

Padda  oryzivora  [Java  sparrow]  is  a  very  suitable  species  for  the  study  of  the 
hsematozoa  of  birds :  it  is  easily  obtained  from  bird  dealers  and  in  three  cases  out 
of  four  in  birds  recently  imported  from  Indo-China  the  blood  contains  H.  danilewskyi 
(Laveran). 

SECTION  II.— THE  GENUS   H^JMOGREGARINA. 

The  blood  parasites  of  the  cold-blood  vertebrata  (fish,  tortoises,  crocodiles, 
pythons  and  all  kinds  of  snakes,  frogs,  salamanders,  tritons,  etc.)  belong  to 
the  genus  H cemogregarina  (Laveran,  Mesnil)  with  which  must  be  included 
the  genus  Drepanidium  of  Lankester  and  the  genus  Danilewskya  of  Labbe. 
These  haematozoa  are  very  numerous  and  some  sixty  species  have  been 
described. 

It  is  now  known  that  several  species  of  the  genus  are  also  parasitic  in  the 
mammalia  (Gerboises,  etc.).  S.  P.  James  has  described  a  parasite,  Leuco- 
cytozoon  canis,  inhabiting  the  white  blood-cells  of  dogs  in  India,  which  should 
apparently  be  included  in  the  genus  H  cemogregarina. 

1.   Hremogregarina  stepanowi. 

This  Hsematozoon  was  discovered  by  Danilewsky  in  the  common  tortoise 
(Cistudo  europcea)  in  which  it  is  a  very  common  parasite  especially  of  the 
adult  tortoise  and  particularly  in  spring  and  summer. 

I.  Laveran  describes  two  forms  of  the  parasite  in  the  blood  of  the  peripheral 
circulation : 

(i)  Reniform,  intra- corpuscular  parasites  measuring  10-14//,  long,  with  rounded 
extremities  and  granular  protoplasm  with  a  nucleus  near  the  centre,  but  containing 
no  pigment.  In  the  fresh  condition,  the  nucleus  appears  as  a  clear,  rounded  or 
oval  space  which  stains  more  deeply  than  the  cytoplasm  with  methylene  blue.  This 
form  of  the  parasite  predominates  in  the  blood  of  some  tortoises. 

(ii)  Worm-like  parasites  situated  within  the  red  cells  and  nearly  always  folded 
upon  themselves.  This  form  is  derived  from  that  just  described  in  the  following 
manner : — When  a  reniform  parasite  has  attained  a  length  of  about  10/z  it  gives 
origin  to  a  segment  which  is  folded  back  upon  the  original  parasite  and  gradually 
increasing  in  length  gives  to  it  the  worm-like  appearance. 

The  vermicules  thus  folded  upon  themselves  measure  15-18/z  long  ;  one  segment 
has  a  swollen  extremity  while  the  other  is  pointed  :  the  nucleus  is  generally  seen 
at  the  bend  and  is  sometimes  compact,  sometimes  elongated,  and  sometimes  divided 
into  two,  the  two  portions  being  connected  by  a  fine  thread  of  protoplasm  (en  besace) ; 
more  uncommonly  it  consists  of  two  distinct  parts.  It  is  especially  well  seen  in 
stained  preparations.  The  vermicules  are  never  pigmented. 

If  the  blood  be  fixed  immediately  it  leaves  the  body  only  intra- corpuscular  vermi- 
cules will  be  seen,  but  if  the  blood  be  kept  for  an  hour  or  so,  free  motile  vermicules 
will  be  found.  These  movements  are  very  active  and  varied,  and  during  progression 
it  will  often  be  noted  that  an  annular  constriction  seems  to  form  round  the  anterior 
end  of  the  parasite  and  pass  in  a  peristaltic  wave  like  a  series  of  rings  towards  the 
posterior  end. 

II.  The  reproduction  forms  of  the  parasite  are  found  in  the  bone  marrow  (Danilew- 
sky) but  especially  in  the  liver  (Laveran). 

According  to  Laveran,  the  forms  corresponding  to  the  reproduction  phase  consist 
of  ovoid  parasites  measuring  10-18//,  long,  at  first  intra-corpuscular  and  later  free : 
they  contain  granules  of  chromatin  staining  with  methylene  blue  and  several  nuclei 


784  ILEMOGREGARINA  RANARUM 

arranged  in  groups  of  three  or  four  at  each  extremity.  Later,  the  contours  of  the 
embryonic  parasites  appear,  the  ovoid  body  divides  either  regularly  or  splits  up  like 
the  staves  of  a  barrel.  The  embryonic  parasites,  which  may  be  either  free  or  con- 
tained in  the  red  cells,  are  elongated,  sometimes  slightly  bent,  swollen  at  one  end 
and  pointed  at  the  other  :  the  nucleus  is  situated  at  the  swollen  end.  The  free 
parasites  are  endowed  with  movement,  thus  enabling  them  to  penetrate  the  red 
cells. 

Reproduction  forms  are  also  found  but  more  rarely,  in  scrapings  from  the  spleen. 

The  reproduction  forms  of  H.  stepanowi  described  by  Laveran  which  represent 
the  endogenous  method  of  reproduction  of  the  parasite  are  very  rarely  seen,  and 
this  may  explain  the  slight  pathogenic  properties  of  the  parasite.  The  exogeneous 
method  of  reproduction  was  for  a  long  time  unknown  :  the  disease  is  not  transmitted 
directly  from  infected  to  non-infected  animals  and  infected  tortoises  do  not  excrete 
a  parasite  capa.ble  of  living  outside  the  body.  According  to  Siegel  H.  stepanowi 
has  a  second  host  in  a  leech  (Placobdella  catenigera  vel  Hcementaria  cof.tata).  In  the 
villi  of  the  hind  gut  of  this  leech  Siegel  found  the  microgamete  stage  and  oocysts 
resulting  from  the  fertilization  by  microgametes  of  the  serpentine  parasites  found  in 
the  blood  of  the  tortoise.  These  oocysts  pass  into  the  blood  spaces  and  thence  into 
the  heart  of  the  leech.  In  the  cesophageal  glands  Siegel  found  spirilliform  bodies 
which  are  probably  sporozoi'tes  capable  of  infecting  other  leeches  :  these  structures 
have  also  been  found  in  the  cesophageal  glands  of  the  embryo  leeches  during  vitelline 
nutrition  ;  it  is  probable  therefore  that  the  egg  itself  becomes  infected. 

Methods  of  examination.  —  Laveran  recommends  the  following  technique. 

(a)  Blood.  —  Blood  may  be  obtained  by  cutting  the  end  of  the  tail  and 
should  be  examined  in  fresh  preparations  as  well  as  after  fixing  and  staining 
(eosin  and  methylene  blue  method,  ride  infra). 

(b)  Tissues.  —  Sections    give    poor    results.      The    following    method    is 
recommended. 

1.  Prepare  a  thin  film  on  a  cover-glass  with  the  tissue  to  be  examined. 

2.  Before  drying  place  the  film  for  30  minutes  in  a  watch-glass  containing 
a  saturated  solution  of  picric  acid.     Wash  in  water. 

3.  Stain  for  6-12  hours  in  the  following  mixture,  which  must  be  freshly 
prepared. 

Saturated  aqueous  solution  of  methylene  blue,  2  c.c. 

Distilled  water,    -          -          -          -          -  -  4    ,, 

1  per  cent,  aqueous  solution  of  eosin,      -  8  drops. 

4.  Wash  in  water,  dehydrate  rapidly  in  absolute  alcohol  and  mount  in 
balsam. 

Laveran  points  out  that  in  these  investigations  care  must  be  taken  to  avoid  mis- 
taking for  parasites  the  nuclei  of  nucleated  red  cells,  the  unstainable  granulations 
of  the  red  cells  of  certain  fish,  chromatin  granules  which 
become  detached  from  the  nuclei  of  red  cells  when  the 


•^^  blood  is  badly  fixed,  and  spherical  granulations  found 

^^J^  Jfe    in  the  red  cells  of  various  chelonians  which  stain  deep 


Jfc    in  the  red  cells  of  various  ch 
I    violet  by  Laveran's  method. 


2.  HoQmogregarina  ranarum. 
Drepanidium  ranarum. 

According  to  Labbe  two  species  of  Drepanidium 
— Drepanidium  princeps  and  Drepanidium  monilis 
are  found  in  the  frog  (Rana  esculenta),  but  Laveran 
considers  that  these  two  forms  really  represent  a 
single  species,  H  cemogregarina  ranarum.     The  para- 
site can  only  be  found  during  the  summer  and 
early  autumn  months, 
(i)  In  the  blood  the  adult  parasite  has  the  appearance  of  a  vermicule,  12-15/x  long, 
which  exhibits  active  and  varied  movement.     In  the  resting  state  the  anterior 


BLEMOGREGARINA  LACERTARUM 


785 


extremity  is  rounded  and  the  posterior  pointed,  but  when  in  motion  the  anterior 
end  also  becomes  pointed  and  so  enables  the  parasite  to  penetrate  the  red  cells ; 
and  further  during  movement  one  or  two  constrictions  may  be  seen  which  beginning 
at  the  anterior  end  seem  to  slide  towards  the  posterior  end  in  a  sort  of  peristaltic  wave. 
The  nucleus  is  situated  about  the  centre  of  the  parasite  and  at  the  posterior  end  there 
is  often  seen  a  structure  of  variable  appearance  which  probably  represents  the 
debris  of  the  red  cell  in  which  the  Drepanidium  developed  or  of  the  membrane 
which  enveloped  the  intra- corpuscular  parasite. 

The  young  intra- corpuscular  parasite  is  represented  by  a  small  nucleated  cell 
with  granular  protoplasm,  of  variable  shape,  measuring  4-8/a  in  its  longest  diameter. 


FIG.  380. — Httmogregarina  ranarum,  (After  Laveran.)  1,  Endo-corpuscular  parasite  : 
2,  free  heemogregarine  :  3,  free  hsemogregarine  with  flagellum  :  4,  Haemogregarine  fixed 
and  stained  :  5,  Haamogregarine  with  constriction  ;  6,  Reproduction  forms. 

As  the  parasite  grows  it  becomes  longer  and  is  occasionally  bent  upon  itself.  It 
sometimes  happens  that  there  may  be  two  parasites  in  the  same  red  cell  and 
leucocytes  are  also  at  times  infected. 

(ii)  Reproduction  forms  are  never  found  in  the  blood  of  the  peripheral  circulation  : 
for  the  study  of  this  phase  of  the  life  history  films  must  be  prepared  from  the  spleen. 

The  parasites  are  numerous  in  the  spleen  even  when  very  few  can  be  found  in 
the  blood-stream.  Reproduction  forms  consist  of  spherical  or  irregular  cells  4-8/x 
in  diameter  each  containing  two  to  six  chromatin  masses  which  stain  deep  violet 
with  haematein.  They  are  very  similar  to  those  of  H  cemogregarina  stepanowi  and 
represent  an  endogenous  phase  of  reproduction  (Laveran).  According  to  Billet, 
exogenous  reproduction  by  sporogony  takes  place  in  a  leech  of  the  genus  Helobdella. 

Methods  of  examination. — Blood  is  obtained  by  pricking  a  toe.  In  other 
details  the  technique  is  the  same  as  described  under  H  cemogregarina  stepanowi. 

3.   Htemogregarina  lacertarum. 

Hsematozoa  are  frequently  found  in  the  blood  of  lizards  (Lacerta  viridis, 
L.  agilis  and  other  species)  and  several  species  have  been  described  by  Danilew- 
sky,  Chalachnikoi?  and  by  Labbe, — H  cemogregarina  lacertarum,  Danilewskya 
lacazei,  etc. 

The  young  intra- corpuscular  parasites  are  rounded  or  oval  nucleated  cells,  but 
they  afterwards  elongate  to  form  vermicules  measuring  about  W/JL  long  and  more 
or  less  bent  upon  themselves.  The  parasite  is  set  free  by  the  destruction  of  the 
infected  red  cells  and  then  shows  active  gregarinoid  movement,  sometimes  accom- 
panied by  swellings  and  constrictions  passing  like  waves  along  the  body.  Repro- 
duction forms  are  not  found  in  the  blood  of  the  peripheral  circulation. 

Methods  of  examination. — Blood  can  be  obtained  by  cutting  the  tip  of  the 
tail.  Films  should  be  prepared  and  stained  in  the  manner  indicated  above. 


3D 


CHAPTER   LIX. 
THE  INTRA-CORPUSCULAR  tt/EMATOZOA  (continued). 

Section  III. — The  genus  Piroplasma,1  p.  786. 
Introduction. 

1.  Piroplasma  bigeminum,  p.  787. 

Morphology  and  method  of  multiplication,  p.   787.     Methods  of  examina- 
tion, p.  790.     Immunity,  p.  791. 

2.  Piroplasma  ovis,  p.  791.     3.  Piroplasma  canis,  p.   791.     4.  Piroplasma  equi, 
p.  792.     5.  Piroplasma  pithed,  p.  793. 

Section  IV. — The  genus  Theileria,  p.  793. 
Theileria  parva,  p.  793. 

BLOOD  parasites  of  the  genus  Piroplasma  (Patton  and  Laveran)  are  the 
cause  of  disease  in  cattle,  sheep,  horses,  dogs,  monkeys  and  possibly  other 
animals. 

The  life  history  of  the  Piroplasmata  is  still  only  imperfectly  understood. 
These  parasites  have  been  investigated  by  Koch  and  Klein,  Nuttall  and 
Graham-Smith,  Kinositha,  Miyajima  and  others,  but  there  is  a  want  of 
agreement  in  the  conclusions  which  these  observers  draw  from  their  observa- 
tions. There  is,  however,  no  doubt  but  that  reproduction  takes  place  both 
by  schizogony  and  by  sporogony.  The  schizonts,  which  may  either  be  intra- 
corpuscular  or  free  in  the  plasma,  are  amoeboid  and  rounded  or  annular  and 
by  a  process  of  budding  give  rise  to  rounded  merozoiites.  The  sporonts  are 
intra-corpuscular  and  pyriform  ;  they  contain  a  nucleus  and  a  blepharoplast 
and  multiply  asexually  by  longitudinal  division  in  the  blood  ;  the  forms 
resulting  from  division  may  remain  attached  by  their  pointed  ends  and 
undergoing  further  division  give  rise  to  star-shaped  forms  or  rosettes 
(cf.  p.  788). 

Sporogony  occurs  in  the  alimentary  canal  of  ticks  (and  perhaps  also  of 
some  biting  flies  and  mosquitos).  According  to  Koch  and  Klein  the  sporonts 
which  are  set  at  liberty  in  the  stomach  of  the  insect  can  be  differentiated  into 
spherical  macrogametes  and  cuneiform  microgametes.  The  fertilized  macro- 
gamete  (zygote)  assumes  a  vermicular  form,  is  motile  and  becomes  club- 
shaped  (ooJcinete). 

Schaudinn,  having  noticed  forms  resembling  Trypanosomes  in  animals 
infected  with  piroplasmata,  put  forward  the  hypothesis  that  the  Piroplasmata 
should  be  grouped  with  the  Trypanosomata.  Miyajima  observed  large 
flagellated  parasites  similar  to  Trypanosomes  in  broth  cultures  sown  with 
the  blood  of  Japanese  oxen  affected  with  piroplasmosis  (P.  parvum  [ Theileria 

1  Synonyms  : — Hcematococcus,  Babes ;  Babesia,  Starcovici ;  Pyrosoma,  Smith  and  Kil- 
bourne ;  Ixodioplasma,  Schmidt. 


THE   PARASITE   OF   TEXAS   FEVER  787 

parva]),  and  this  observation  would  tend  to  confirm  Schaudinn's  hypothesis  ; 
but  Martini  and  Crawley  have  shown  that  the  flagellated  parasites  seen  by 
Miyajima  are  not  Piroplasmata  and  that  he  was  dealing  with  a  double 
infection  with  two  distinct  parasites. 

1.  Piroplasma  bigeminum. 

Synonyms. — Pyrosoma  bigeminum.     Babesia  bigemina. 

Piroplasma  bigeminum  was  the  name  given  by  Smith  and  Kilbourne  to 
the  parasite  causing  Texas  fever  in  cattle.  Babes  had  previously  recorded 
the  presence  of  the  same  hsematozoon  in  the  haemoglobinuria  of  cattle  associ- 
ated with  bacteria.  The  parasite  has  also  been  found  in  cattle  suffering 
from  hsemoglobinuria  by  Krogius  and  von  Hellens  in  Finland,  by  Theiler  in 
South  Africa,  in  cattle  in  the  Crimea  affected  with  bovine  malaria  by  Laveran 
and  Nicolle,  in  Argentina  in  the  disease  of  cattle  known  as  tristeza,  and  in 
France  in  le  mal  de  brou. 

Symptoms  and  lesions. — Bovine  piroplasmosis  assumes  either  an  acute,  or  a 
subacute  or  attenuated  form.  The  acute  form  of  the  disease  is  most  common  in 
summer  and  is  generally  fatal :  the  temperature  is  raised,  the  urine  is  blood-stained 
and  often  contains  albumen,  the  animal  loses  its  appetite  and  is  constipated, 
rumination  is  suspended,  the  blood  becomes  fluid  and  very  pale- coloured,  the  animal 
emaciates  and  sometimes  shows  nervous  symptoms  (delirium,  paralyses)  and  death 
takes  place  within  a  few  days :  in  a  small  percentage  of  cases  the  animal  recovers 
but  relapses  are  of  frequent  occurrence. 

The  subacute  form  of  the  disease  occurs  more  commonly  in  autumn  and  may  be 
overlooked  if  the  blood  be  not  examined.  There  is  no  haemoglobinuria,  the  fever 
is  not  so  marked  and  the  symptoms  are  generally  far  less  severe. 

Post  mortem. — In  animals  dead  of  Texas  fever  sub-cutaneous  ecchymoses  are 
frequently  found,  the  spleen  is  considerably  enlarged,  the  perirenal  tissues  are 
cedematous  and  the  kidneys  enlarged  and  congested  :  patches  of  pulmonary  hepatiza- 
tion  are  also  sometimes  found.  Many  of  the  red  cells  of  the  blood  are  enlarged  and 
their  number  much  diminished  :  the  leucocytes  are  sometimes  increased  in  number. 

^Etiology. — The  researches  of  Smith  and  Kilbourne,  of  Koch  and  of  Theiler  show 
that  the  disease  is  spread  by  ticks  [Boophilus  annulatus,  B.  dugesi,  B.  decoloratus^ 
Ixodes  ricinus  and  H cemaphysalis  punctate*,].  If  all  the  ticks  be  removed  from  an 
animal  before  it  is  imported  to  a  "clean"  area,  there  is  no  risk  of  the  animals  in  the 
latter  contracting  the  disease.  If  ticks  be  taken  from  cattle  suffering  from  Texas- 
fever  and  placed  01  pasture  where  healthy  beasts  are  grazing  the  latter  soon  show 
symptoms  of  the  disease.  The  infection  however  is  not  conveyed  by  the  tick  which 
sucked  the  infected  blood,  but  by  the  next  generation,  the  parasite  being  transmitted 
from  one  generation  of  ticks  to  the  next  through  the  eggs  (Smith  and  Kilbourne, 
Koch  and  others).  Female  ticks  after  feeding  on  infected  cattle  fall  to  the  ground, 
lay  their  eggs  and  die :  the  larvae  hatched  from  these  eggs  will  infect  the  animals 
upon  which  they  become  parasitic. 

Morphology  and  method  of  multiplication. 

1.  Appearance  in  the  blood.— In  almost  all  cases  Laveran  and  Nicolle  have 
found  the  piroplasm  within  the  red  cells  of  the  peripheral  circulation  in  two 
chief  forms  : 

1.  Small,  spherical  or  oval  parasites.  In  stained  preparations  a  mass  of 
chromatin  generally  consisting  of  two  unequal  parts — the  nucleus  and 
Uepharoplast — can  be  made  out,  situated  as  a  rule  near  the  periphery  of  the 
parasite.  The  smallest  of  these  forms  does  not  measure  more  than  about 
I//,  in  diameter.  In  the  largest  forms  the  nucleus  elongates  and  divides  into 
two,  the  two  parts  remaining  attached  for  a  time  but  afterwards  separating 
and  passing  to  the  opposite  ends  of  the  parasite,  after  which  the  proto- 
plasm divides.  This  represents  the  schizogonous  —  asexual  —  method  of 
reproduction. 


788  THE  INTRA-CORPUSCULAR  ILEMATOZOA 

2.  Pyriform  parasites  measuring  2 '5-3 '5^  long  and  occurring  in  pairs,  the 
narrow  end  of  each  being  continuous  with  or  contiguous  to  the  tapering  end 
of  the  other — hence  the  name  bigeminum.  Sometimes  two  entirely  separate 
pyriform  parasites  are  seen,  in  which  case  the  pointed  ends  are  often  turned 
away  from  each  other.  In  stained  preparations  two  masses  of  chromatin 


FIG.  381. — Piroplasma  bigeminum.     Blood  from  a  cow.      x  1000. 

can  be  seen  in  the  larger  end  of  the  pear-shaped  parasite — a  rounded  or  oval 
nucleus  and  a  blepharoplast — and  the  pointed  end  often  shows  scattered 
granules  of  chromatin. 

In  some  of  the  free  forms  the  pointed  end  of  the  parasite  is  prolonged  into 
a  pseudo-flagellum  terminating  in  a  point  and  staining  with  difficulty  (Lig- 
nieres,  Fantham). 

In  the  blood  of  the  peripheral  circulation  the  pyriform  bodies  are  much 
more  numerous  than  the  round  or  oval  forms.  They  represent  sexual  sporonts, 
development  taking  place  in  the  stomachs  of  ticks  (Doflein  and  Liihe). 

In  the  blood,  these  pyriform  parasites  multiply  asexually  by  longitudinal 
division,  the  chromatin  dividing  first :  the  infected  red  cell  then  contains 
two  parasites  :  occasionally  these  two  parasites  divide  again  and  the  red 
cell  then  contains  four  pyriform  parasites.  The  occurrence  of  four  parasites 
in  one  cell  may  also  be  accounted  for  by  supposing  that  it  was  originally 
infected  with  two  parasites. 

[Nuttall  and  Graham-Smith 1  from  a  prolonged  study  of  the  living  parasite 
of  canine  piroplasmosis  and  a  comparative  study  of  stained  films  of  P.  canis, 
P.  bigeminum  (P.  bovis)  and  P.  pithed  have  established  the  fact  that  these 
three  species  multiply  in  precisely  the  same  manner,  namely  by  a  peculiar 
process  of  budding  by  which  a  single  amoeboid  body  usually  gives  rise  to  two 
pyriform  parasites. 

[A  free  pyriform  parasite  enters  a  normal  red  blood  corpuscle  and  after  a 
time  assumes  a  rounded  form,  grows,  and  becomes  actively  amoeboid.  The 
amoeboid  parasite  then  protrudes  two  symmetrical  bud-like  processes,  which 
rapidly  grow  and  become  pear-shaped  ;  the  protoplasm  of  the  parasite 
flows  into  these  "  buds  "  and  the  body  consequently  becomes  smaller  until 
it  is  represented  by  a  minute  mass  to  which  the  pyriform  bodies  are  attached. 
This  minute  mass  ultimately  disappears  and  two  mature  pyriform  parasites 
are  left,  joined  for  a  time  by  a  filament  which  finally  ruptures  leaving  them 
free.  After  a  variable  length  of  time  the  parasites  escape  from  the  corpuscles 

1  Journal  of  Hygiene  IV.,  V.,  VI.,  VII.,  and  Parasitology  I. 


THE   PARASITE   OF  TEXAS   FEVER 


789 


and  the  moment  they  escape  attack  fresh  corpuscles  and  repeat  the  same 
method  of  multiplication. 


showing  the  usual  mode 

of  multiplication 

of 

Piroplasma  canis 

in  the  circulating  blood. 


FIG.  382. — Cycle  showing  the  usual  mode  of  multiplication  of  Piroplasma 

canis  in  the  circulating  blood  (Nuttall). 

From  the  Journal  of  Hygiene  (Cambridge  University  Press)  by  permission  of 
Professor  G.  H.  F.  Nuttall,  F.R.S. 

[A  pyriform  parasite  possesses  a  dense  mass  of  chromatin,  usually  situated 
at  the  pointed  extremity,  and  a  secondary  mass  of  loose  chromatin  extending 


790  THE  INTRA-CORPUSCULAR  KLEMATOZOA 

towards  the  blunt  end.  When  the  parasite  becomes  rounded  the  chromatin 
gradually  fuses  into  one  mass,  which  subsequently  divides  and  forks  in  a 
peculiar  manner  so  as  to  give  rise  to  a  Y-shaped  chromatin  figure  ;  the  two 
processes  are  thin,  and  protrude  into  the  two  small  buds  and  indicate  coming 
division.  As  the  buds  grow  in  size,  the  main  mass  of  chromatin  divides  and 
all  the  chromatin  passes  into  the  two  pyriform  bodies. 

[Moreover  an  amoeboid  intra-corpuscular  parasite  may  divide  in  the 
.amoeboid  stage  into  two  parasites  and  the  two  daughter  cells  give  rise  inde- 
pendently and  perhaps  simultaneously,  to  two  pairs  of  pyriform  parasites 
in  a  similar  manner  to  that  described.] 

2.  Appearance  in  films  prepared  from  the  spleen. — Free  parasites  are  much 
more  numerous  in  the  spleen  than  in  the  blood  of  the  peripheral  circulation. 
The  small  rounded  or  oval  forms  predominate  and  undergo  multiplication — 
this  organ  being  the  main  site  of  endogenous  reproduction  (schizogony). 
These  small  forms  being  endowed  with  powers  of  amoeboid  movement  are 
able  to  penetrate  the  red  cells  (Laveran  and  Mesnil). 

Appearance  in  ticks. — The  changes  taking  place  in  Piroplasma  bigeminum 
in  the  stomachs  of  ticks  after  the  latter  had  fed  on  infected  animals  has  been 
followed  by  Koch.  As  has  already  been  stated  it  is  in  the  stomachs  of  certain 
species  of  ticks  that  the  piroplasma  undergoes  sexual  (sporogonous)  multi- 
plication. About  12  or  20  hours  after  the  infected  meal  it  is  not  uncommon 
to  find  the  pyriform  parasites,  which  have  now  left  the  red  cells,  changed 
into  spherical  bodies  each  having  twelve  to  twenty  delicate  straight  pro- 
longations arranged  round  it  like  rays  round  a  star.  These  ray-like  processes 
subsequently  disappear  while  the  spherical  bodies  increase  in  size  and  later 
large  club-shaped  structures  (ookinetes)  are  seen,  not  only  in  the  digestive 
tube  but  also  in  the  eggs. 

Methods  of  examination. 

Microscopical  examination.— The  blood  from  an  infected  animal  may  be 
examined  either  in  the  fresh  condition  or  after  drying. 
Laveran  and  Nicolle  give  the  following  directions  : 

1.  Fix  the  films  at  110°  C.  for  a  few  minutes  and  then  in  a  saturated  aqueous 
solution  of  perchloride  of  mercury  for  1  minute. 

2.  Stain  for  1-2  hours  in  Laveran's  eosin-methylene-blue  solution,  wash, 
treat  with  tannin  and  proceed  as  described  at  p.  772.     Before  mounting  in 
balsam,  make  certain  that  the  preparation  is  not  stained  too  deeply  :   should 
this  be  the  case,  decolourize  in  absolute  alcohol.     The  nuclei  of  the  parasites 
should  be  stained  violet-red. 

Cultures. — A  culture  of  P.  bigeminum  may  be  obtained  if  some  blood  rich 
in  parasites  be  sown  in  citrated  blood  or  in  serum  rich  in  haemoglobin  and 
incubated  at  37°  C.  (Lignieres).  Growth  appears  in  about  a  fortnight.  The 
piroplasma ta  become  rounded,  leave  the  red  cells  and  lose  their  nuclei :  then 
the  nucleus  is  formed  anew  and  divides  into  from  two  to  five  small  spherical 
bodies  surrounded  by  protoplasm,  constituting  spores.  These  in  their  turn  give 
origin  to  new  spherical  bodies.  Pyriform  bodies  are  never  seen  in  cultures. 

Experimental  inoculation. — Cattle  are  the  only  domestic  animals  susceptible 
to  infection.  Infection  of  cattle  can  be  produced  experimentally  by  inocu- 
lating infected  blood  beneath  the  skin,  into  the  muscles  or  into  the  peripheral 
circulation.  The  success  of  the  experiment  will  depend  upon  the  number  of 
parasites  in  the  blood  inoculated  ;  when  it  is  rich  in  parasites  a  dose  of 
01-0-05  c.c.  will  be  sufficient  to  produce  infection,  but  it  may  be  necessary 
to  use  as  much  as  1,  2  or  even  10  c.c. 

Cattle  cannot  be  infected  by  the  alimentary  canal. 


PIROPLASMOSIS   OF  SHEEP  791 

Immunity. 

Bovine  piroplasmosis  does  not  recur  in  the  same  animal  and  cattle  which 
recover  from  a  first  attack  of  the  disease  are  permanently  immune.  The 
serum  of  such  animals  has  neither  therapeutic  nor  prophylactic  properties 
(Nicolle  and  Adil-Bey).  Lignieres  obtained  promising  results  by  vaccinating 
cattle  with  an  attenuated  piroplasma. 

In  the  blood  of  immunized  animals  the  piroplasma  assumes  a  different 
form  (rings  and  small  rods)  from  that  presented  by  the  parasite  in  animals 
suffering  from  the  disease.  The  inoculation  of  blood  containing  these  parasites 
into  an  healthy  animal  produces  an  illness  which  may  terminate  fatally 
(Theiler). 

2.  Piroplasma  ovis. 

Synonym. — Babesia  ovis. 

The  piroplasma  infecting  sheep  was  first  described  by  Babes  in  the  disease 
known  as  Carceag,  in  Roumania.  It  was  subsequently  found  by  Bonome  in 
an  epizootic  near  Padua  and  has  been  studied  by  Laveran  and  Nicolle  (in  an 
epizootic  in  Constantinople)  and  by  Motas  (in  Koumania).  The  parasite  has 
also  been  found  in  Bulgaria,  Italy,  France,  South  Africa  and  India. 

Sheep  piroplasmosis  occurs  in  two  forms — a  severe  and  fatal  form  (with  anaemia, 
prostration  and  haemoglobinuria)  and  a  mild  form  often  not  diagnosed  which  ter- 
minates in  recovery.  One  attack  confers  immunity.  In  sheep  dead  of  piroplasmosis 
the  tissues  are  oedematous,  the  blood  pink  and  fluid,  the  spleen  and  the  lymphatic 
glands  enlarged :  numerous  parasites  will  be  found  in  the  blood  and  spleen. 

Appearance  in  the  blood. — The  parasites  measure  1-1 '5/x.  in  diameter  ;  they 
are  rounded  or  elongated,  non-pigmented,  generally  contained  within  the 
red  cells,  and  have  a  rounded  karyosome  situated,  as  a  rule,  near  the  periphery. 
Some  of  the  intra-corpuscular  parasites  will  be  seen  to  be  actively  multiply- 
ing :  the  karyosome  elongates  and  then  splits  into  two,  and  division  of  the 
protoplasm  follows.  The  double  and  pyriform  parasites  are  as  a  rule  free 
but  may  occasionally  be  found  in  the  red  cells.  Generally  speaking  there  is 
not  more  than  one  parasite  in  a  red  cell. 

Appearance  in  films  from  the  spleen. — The  parasites  are  more  numerous  in 
the  spleen  than  in  the  blood  of  the  peripheral  circulation  but  are  similar  in 
appearance,  though  they  are  often  a  little  larger  and  dividing  forms  are 
more  common. 

Microscopical  examination. — The  technique  is  the  same  as  in  the  case  of 
Piroplasma  bigeminum. 

^Etiology. — Piroplasma  ovis  is  transmitted  by  the  sheep  tick  (Rhipicephalus 
bursa).  As  in  other  piroplasmoses  it  is  the  progeny  of  the  ticks  which  have  sucked 
the  blood  of  infected  animals  which  transmit  the  disease.  These  daughter  ticks 
are  only  capable  of  inoculating  the  parasite  when  they  have  reached  maturity 
(Motas).  As  a  rule  one  attack  confers  immunity. 

3.  Piroplasma  canis. 

Canine  piroplasmosis  is  a  common  disease  in  France  (Leblanc,  Nocard)  : 
it  occurs  also  in  Senegal  (Marchoux),  in  South  Africa  (Theiler :  malignant 
jaundice,  biliary  fever  of  dogs),  in  Tonkin  (Mathis),  in  Japan  (Kinositha), 
etc. 

^Etiology. — Canine  piroplasmosis  is  propagated  by  dog  ticks  [(H cemapJiysalis 
leachi  in  Africa  :  Rhipicephalus  sanguineus  in  India)  ]  (Nuttall,  Lounsbury).  The 
disease  is  transmitted  by  the  offspring  of  those  ticks  which  have  sucked  the  blood  of 
infected  animals  but  only  when  the  daughter  ticks  are  full-grown.  A  first  attack 


792  THE   INTRA-CORPUSCULAR   H^EMATOZOA 

confers  immunity.     The  blood  of  immunized  animals  contains  the  parasite  and  is 
capable  of  setting  up  a  fatal  disease  in  a  non-immune  animal. 

Appearance  in  the  blood.— The  parasites,  numerous  in  the  acute  form,  are 
somewhat  rare  in  the  chronic  form  of  the  disease.  This  species,  like  P. 
bigeminum,  is  found  in  the  blood  in  two  forms  : 

1.  The    round,  amoeboid  forms — sometimes    small    and    free,    sometimes 
discoid  or  annular — contained  in  the  red  cells  divide  by  budding  (Kinositha)  : 
the  annular  forms  consist  of  a  ring  of  protoplasm  enclosing  a  central  vacuole  ; 
the  protoplasm  contains  a  chromatin  mass  divided  into  nucleus  and  blepharo- 
plast  (Liihe). 

2.  The  pear-shaped  forms  appearing  particularly  during  the  second  period 
of  the  infection  correspond,  according  to  Kinositha,  to  sexually  differentiated 
sporonts ;   but  in  the  blood  they  may  multiply  by  binary  longitudinal  divi- 
sion.    Some  parasites  have  one  or -two  flagella  (Breinl,  Kinositha).     The 
pyriform  bodies  are  free  or  intra-corpuscular  :   some  of  the  red  cells  contain 
as  many  as  eight  parasites. 

[Cf.  Nuttall  and  Graham-Smith's  description  (p.  788).] 

Development  in  the  tick. — Christophers,  following  the  development  of  the 
parasite  in  Rhipicephalus  sanguineus,  describes  a  sporogonic  development 
as  taking  place  in  the  alimentary  canal  of  the  tick  resulting  in  the  formation 
of  vermicular  elements  which  pass  into  the  ovaries  of  the  adult  tick  and  into 
the  embryonic  tissues  of  the  nymph,  there  forming  a  rounded  mass,  or  zygote, 
which  in  turn  gives  origin  to  sporoblasts  and  these  to  sporozo'ites. 

Cultures. — In  vitro,  Kleine  has  observed  modifications  of  the  parasite 
without  true  multiplication,  in  diluted  defibrinated  blood.  The  blood  is 
collected  from  infected  dogs  some  hours  before  death,  defibrinated  and  mixed 
with  an  equal  volume  of  normal  saline  solution  and  incubated  at  25°  C.  to 
27°  C. 

Under  these  conditions,  club  forms  with  radiating  processes  are  seen  after 
about  12  hours  similar  to  those  described  by  Koch  in  the  case  of  P.  bigeminum  : 
these  bodies  exhibit  very  distinct  amoeboid  movement.  They  assume  a 
rounded  form  towards  the  second  day,  the  radiating  processes  become 
obliterated  and  the  parasites  then  themselves  disappear. 

Methods  of  detection. — The  technique  is  similar  to  that  for  Piroplasma 
bigeminum.  Nocard  advises  fixing  the  blood  films  in  absolute  alcohol  and 
staining  with  carbol-thionin. 

To  facilitate  the  diagnosis  in  cases  in  which  the  parasites  are  few  in  number 
and  where  microscopical  examination  has  failed  to  reveal  their  presence, 
Nocard  recommends  injecting  into  a  young  dog  sub-cutaneously  or  intra- 
venously, 5-10  c.c.  of  the  blood  of  the  dog  under  examination.  If  it  be  a 
case  of  piroplasmosis  the  inoculated  animal  as  a  rule  develops  an  acute  piro- 
plasmosis,  and  after  the  third  to  the  fifth  day  the  parasites  multiply  in  its 
blood. 

4.  Piroplasma  equi. 

Equine  piroplasmosis  is  a  common  disease  in  the  Transvaal,  where  it 
has  been  studied  by  Theiler :  it  is  very  similar  to  "  red-water  "  in  cattle. 
The  parasite  was  discovered  by  Laveran  ;  it  is  smaller  than  P.  bigeminum, 
and  as  generally  seen  is  round  or  oval,  pyriform  bodies  being  very  rare.  The 
disease  is  transmitted  by  a  tick,  Rhipicephalus  evertsi  (Theiler). 

Piroplasma  equi  has  been  found  in  Madagascar  by  Thiroux  in  horses  suffering 
from  a  chronic  disease  known  by  the  inappropriate  name  of  Osteomalacia. 

Equine  piroplasmosis  is  a  common  disease  in  India  (Patton),  where  it  is 
said  to  be  transmitted  by  biting  flies  or  mosquitos  (Williams). 


THE   PARASITE   OF  EAST  COAST  FEVER  793 

Imported  animals  suffer  from  the  disease  but  animals  born  and  bred  in 
tick-infested  countries  have  an  acquired  or  transmitted  immunity  (Theiler). 
The  blood  of  an  immune  animal  contains  the  parasite  and  will  infect  non- 
immune  animals. 

5.  Piroplasma  pitheci. 

[A  true  piroplasmosis  occurs  in  monkeys  (CercopitHecus)  in  Uganda  and 
was  first  observed  by  P.  H.  Ross.  The  manner  in  which  the  disease  is  trans- 
mitted is  as  yet  unknown  (Nuttall). 

[The  appearance  of  the  parasite  in  the  blood  and  the  mode  of  division  is 
the  same  as  in  P.  bigeminum  and  P.  canis  (p.  788)  (Nuttall  and  Graham- 
Smith).] 

SECTION  IV.— THE   GENUS  THEILERIA. 

[Theileria  parva.1] 

[Rhodesian  fever  of  cattle  (East  coast  fever,  tropical  piroplasmosis)  is  due 
to  an  infection  with  a  blood  parasite  which  resembles  the  parasite  of  red-water 
in  that  it  is  transmitted  by  ticks  (Rhipicephalus  appendiculatus  and  R.  simus 
[and  other  species  of  the  same  genus])  but  differs  from  it  both  in  morphology 
and  in  the  fact  that  it  cannot  be  transmitted  by  inoculation.  ] 

According  to  Theiler,  Rhodesian  fever,  which  is  the  worst  of  all  cattle  diseases, 
may  assume  one  of  two  clinical  types  :  an  acute  rapidly  fatal  form  accompanied  by 
fever,  blood-stained  diarrhoea,  intense  jaundice  and  muscular  twitchings,  and  a 
chronic  form  characterized  by  a  transitory  attack  of  fever  and  jaundice. 

Appearance  of  the  parasite  in  the  blood. — In  the  blood  of  infected  cattle 
three  forms  of  the  parasite  are  found.  In  the  acute  form  of  the  disease  the 
parasites  assume  a  ring  or  bacillary  form  and  not  infrequently  the  one  may 
be  seen  to  change  into  the  other  :  they  exhibit  amoeboid  movement,  and  a 
small  mass  of  chromatin  can  be  made  out.  In  the  chronic  form  of  the  disease 
the  parasite  appears  as  a  non-motile  punctiform  mass  of  chromatin. 

The  parasites  are  very  abundant  in  the  blood  :  in  the  acute  form  of  the 
disease  90  per  cent,  of  the  red  cells  may  contain  them.  Generally  a  few  red- 
water  parasites  are  also  seen,  in  which  cases  the  animals  are  suffering  from  a 
double  infection. 

Appearance  in  ticks. — In  ticks,  Koch  observed  starred  and  rounded  forms, 
similar  to  those  seen  in  the  case  of  Piroplasma  bigeminum. 

Microscopical  examination. — The  technique  is  the  same  as  for  P.  bigeminum. 

Cultures. — Dschunkowsky  and  Liihs  have  observed  multiplication  of  the 
parasite  in  serum  stained  with  haemoglobin  obtained  from  animals  suffering 
from  the  disease. 

Experimental  inoculation. — The  disease  cannot  be  transmitted  by  inocula- 
tion :  even  when  the  blood  inoculated  is  swarming  with  parasites  the  animal 
does  not  contract  the  disease.  Animals  which  have  recovered  from  an  attack 
of  the  disease  are  immune  :  the  parasite  cannot  be  found  in  the  blood  and 
ticks  fed  on  the  blood  do  not  become  infected. 

Theiler  has  recently  described  a  parasite  morphologically  similar  to  the  foregoing 
but  feebly  pathogenic  to  cattle  and  very  easily  inoculable.  This  parasite  is  known 
as  Piroplasma  mutans  (Theiler). 

Piroplasma  mutans  is  very  often  found  in  cattle  in  association  with  P.  bigeminum. 

1  [This  parasite  is  sometimes  regarded  as  belonging  to  the  genus  Piroplasma — Piroplasma 
parvum,  Babesia  parva — but  Bettencourt,  Franca  and  Borges  consider  that  it  differs  so 
widely  from  the  parasites  of  that  genus  that  it  should  be  separated  from  them  and  for 
that  reason  created  the  new  genus  Theileria.  Nuttall  is  of  the  same  opinion.  ] 


CHAPTEK  LX. 

THE   GREGARINIDA. 

[THE  Gregarinida  are  an  Order  of  the  Sub-division  Telosporidia  of  the 
Sporozoa  (see  p.  760).] 

The  Gregarines  are  unicellular  parasites  which  live  in  the  gut  or  body 
cavity  of  the  invertebrata  and  especially  in  the  articulata.  [They  are  wholly 
or  in  part  intra-cellular  parasites  inhabiting  usually  the  epithelial  cells  of 
the  host,  but  never  the  blood  cells.] 

In  the  adult  form  [Trophozo'ite]  they  are  more  or  less  elongated  structures 
measuring  from  10-20/x  to  16  mm.  and  consist  of  a  continuous  outer  membrane 
or  cuticle  (epicyte)  containing  an  ectoplasm,  endoplasm  and  a  nucleus.  The 
body  of  a  Gregarine  may  be  homogeneous  or  segmented  hence  the  two  groups 
monocystida  and  dicystida.  In  the  dicystida  the  segments  are  generally 
unequal  in  size  ;  the  anterior  segment  or  head  is  known  as  the  protomerite 
and  in  some  cases  possesses  an  organ  of  attachment,  the  epimerite ;  the 
posterior  segment  or  body  known  as  the  deutomerite  contains  the  nucleus. 

The  cuticle  or  epicyte  frequently  shows  longitudinal  striae  composed  of 
myonemes.  Transverse  striation  is  less  commonly  seen. 

The  cytoplasm  consists  of  an  ectoplasm  and  endoplasm  the  latter  as  a  rule 
containing  a  large  number  of  chromatic  granules. 

The  nucleus,  usually  single^  is  spherical  or  oval  and  has  several  nucleoli. 

Those  Gregarines  which  have  an  epimerite  remain  attached  for  some  time 
to  an  epithelial  cell  of  the  host  then  by  rupture  of  the  junction  between  the 
epimerite  and  protomerite  the  parasite  becomes  detached  and  is  free. 

The  Gregarines  exhibit  movement,  sometimes  of  translation  and  some- 
times of  flexion :  movements  of  flexion  are  limited  to  the  deutomerite  and 
are  accompanied  by  active  contractions  of  the  protoplasm. 

At  a  given  moment  in  its  life  history  the  Gregarine  becomes  encysted, 
whether  it  has  had  previous  association  with  another  of  its  kind  or  not. 

In  this  association  which  is  not  a  conjugation  but  merely  a  juxtaposition 
two  individuals  become  attached  together — [syzygy] — either  by  their  anterior 
ends  as  is  the  case  with  but  few  exceptions  in  the  Monocystids  or  by  opposite 
ends  as  in  the  Dicystids.  [The  two  associated  individuals  then  generally 
become  surrounded  by  a  common  membrane.]  In  Gonospora  longissima 
Caullery  and  Mesnil  have  shown  that  the  septum  between  the  two  associated 
individuals  is  destroyed  and  that  the  protoplasm  of  the  parasites  fuses. 
Occasionally  a  number  of  individuals  become  attached  end  to  end  forming 
a  chain. 

[After  encystment l  the  nuclei  of  the  two  trophozoltes  or  sporonts  or  gameto- 

t1  See  Professor  Minchin's  description  in  A  Treatise  on  Zoology  edited  by  E.  Ray 
Lankester,  London,  1909.] 


THE   GREGARINIDA 


795 


cytes  undergo  karyokinetic  division  and  the  daughter  nuclei  pass  to  the 
surfaces  of  the  encysted  parasites.  The  cytoplasm  of  each  cell  divides  into 
an  equal  number  of  segments  and  the  segments  collect  around  the  nuclei : 
each  of  these  small  nucleated  masses  of  protoplasm  is  known  as  a  sporoblast 
or  gamete.  A  certain  amount  of  cytoplasm — known  as  the  cystal  residuum — 
remains  unused  and  serves  for  the  nutrition  of  the  gametes.  The  cuticle  of 
each  gametocyte  now  dissolves  ;  the  gametes  exhibit  active  movement  and 
conjugate  in  pairs — a  gamete  from  one  gametocyte  conjugating  notably 
with  a  gamete  from  the  other.  After  uniting  each  pair  of  gametes  becomes 
a  zygote.  The  zygote  becomes  oval  and  secretes  a  chitinous  envelope,  forming 
a  sporocyst.  Within  this  cyst  the  nucleus  of  the  zygote — or  as  it  is  now 
termed  the  sporoplasm — divides  into  two,  then  into  four  and  finally  into 
eight  nuclei  which  take  up  an  equatorial  position  and  become  surrounded 
each  by  a  part  of  the  protoplasm  of  the  sporoplasm.  In  this  way  the  sporo- 
zo'ite  or  falciform  body  is  formed.  In  Monocystis  the  fully  formed  sporozoi'te 
has  a  more  or  less  boat  shape  and  resembles  a  diatom  of  the  genus  Navicella, 
hence  the  name  Pseudonavicella  by  which  Gregarine  spores  are  generally 
known. 

[The  spores  of  Monocystis  do  not  appear  to  be  able  to  develop  further  in 
the  earthworm  but  require  to  be  transferred  to  a  new  host.  It  is  probable 
that  infection  of  a  new  host  takes  place  by  way  of  the  alimentary  canal,  the 
digestive  juices  dissolving  the  wall  of  the  sporocyst  and  setting  free  the  sporo- 
zo'ite  which  is  actively  motile  and  able  to  bore  its  way  through  cells  and 
tissues. 

[When  Gregarines  become  encysted  without  pairing  the  gametocyte  breaks 
up  into  gametes  at  once.  In  these  cases  the  spores  are  smaller  than  those 
produced  from  zygotes.] 


FIG.  383. — Gregarine  of  the  lobster.  On  the  right,  the  adult  form  of  the 
parasite  ;  on  the  left,  the  different  shapes  assumed  by  the  protozoon  in  passing 
from  the  young  amo3boid  to  the  adult  form.  (After  V.  Beneden.) 

From  the  observations  of  Caullery  and  Mesnil  it  appears  that  in  some 
Gregarines  multiplication  takes  place  by  schizogony,  the  intra-cellular  stage 
of  the  life  history  being  prolonged.  In  Gonospora  longissima,  a  parasite  of 
Dodecaceria  concharum,  the  sporozoite  set  free  from  the  spbres  in  the  gut  of 
the  Annelid  host  passes  into  one  of  the  epithelial  cells  lining  the  gut  and  in 
this  cell  gives  origin  to  a  small  spherical  nucleated  body  which  increasing  in 
size  becomes  the  trophozoi'te  ;  and  this  multiplying  by  division  divides  into 
six  or  eight  crescent-shaped  merozoi'tes  arranged  side  by  side  to  form  a 


796  THE   GREGARINIDA 

barrel-shaped  structure.  These  crescent-shaped  merozoites  then  escape  from 
the  cell  host  and  become  sporozoites  which  pass  into  the  body  cavity  of  the 
animal. 

Among  the  best  known  Gregarines  are  Porospora  gigantea  (parasitic  in  the  intes- 
tine of  Astacus  gammarus)  ;  Gregarina  blattarum  (parasitic  in  the  intestine  of  Blatta 
orientalis) ;  Monocystis  tenax  (a  parasite  of  the  earthworm)  ;  Lankesteria  ascidice 
(a  parasite  of  Ciona  intestinalis). 

Technique. — For  the  examination  of  the  Gregarinida,  Wasielewski  recom- 
mends osmic  acid  or  a  saturated  solution  of  perchloride  of  mercury  as  a  fixing 
agent,  and  safranin,  picro-carmine,  gold  chloride  or  silver  nitrate  as  a  stain. 
Bertarelli  advises  fixing  in  acid  perchloride  or  in  Flemming's  solution  and 
staining  in  iron-hsematoxylin  or  Delafi eld's  haeinatoxylin. 


CHAPTER  LXI. 
PARASITES  OF  THE   GENUS  LEISHMANIA. 

1.  Leishmania    donovani,   p.    797.     2.  Leishmania    infantum,   p.   800.     3.  Leish- 
mania tropica,  p.  802.     4.  Other  species  of  Leishmania,  p.  802. 

1.  Leishmania  donovani. 

Synonym. — Piroplasma  donovani. 

IN  1900  Leishman  demonstrated  [in  the  spleen  of  a  man  who  had  been 
invalided  for  and  who  died  of  dum-dum  fever l]  certain  parasitic  forms  which 
he  suggested  might  be  Trypanosomes.  In  1903  Donovan  found  the  same 
parasite  in  the  smears  of  the  spleen  taken  from  patients  dying  of  a  similar 
disease  in  Madras,  and  Bentley  subsequently  found  them  in  patients  suffering 
from  Kala  Azar,  a  very  fatal  disease  found  in  Bengal  and  Assam. 

The  Leishman-Donovan  parasite  was  originally  classified  by  Laveran  and 
Mesnil  with  the  piroplasmata  :  Ross  however  created  for  it  a  new  genus — 
Leishmania.  Marchand  and  Ledingham  consider  that  the  parasite  is  a  Try- 
panosome  while  Rogers  [and  Patton]  group  it  with  the  genus  Herpetomonas. 

Methods  of  detecting  the  parasite.  Its  appearance  in  the  tissues.— The 
parasite  occurs  characteristically  in  the  endothelial  cells  of  the  blood  and 
lymphatic  vessels  :  it  is  present  in  very  large  numbers  in  the  spleen,  liver 
and  bone  marrow,  and  is  also  found  in  the  lungs,  kidneys,  mesenteric  glands 
and  in  the  ulcers  in  the  large  intestine.  In  the  peripheral  blood  it  occurs  in 
small  numbers  within  the  polymorpho-  and  mono-nuclear  leucocytes  and 
very  occasionally  in  the  red  cells.  Its  presence  in  the  blood  is  very  likely  to 
be  missed  so  that  several  preparations  should  be  examined  before  coming  to  a 
negative  conclusion  :  in  the  last  stage  of  the  disease,  however,  and  during 
febrile  attacks  the  parasite  may  be  present  in  large  numbers  in  the  blood- 
stream (Donovan). 

Blood-films  should  be  prepared  and  examined  in  the  first  instance  and 
should  the  parasite  not  be  found  the  spleen  may  be  punctured  and  films 
stained.  Puncture  of  the  spleen,  however,  is  always  a  risky  proceeding :  it 
is  better  to  puncture  the  liver.  Use  a  quite  dry,  sterile  syringe  with  a  fine 
needle  :  the  results  are  more  satisfactory  if  no  blood  be  drawn.  Expel  the 
material  in  the  needle  on  to  slides,  spread  films,  dry,  stain  with  Giemsa's  or 
Leishman's  stain  and  examine  with  an  oil-immersion  lens. 

1  [Dum-dum  fever  is  so  called  from  Dum-dum  an  unhealthy  cantonment  near  Calcutta.  ] 
The  disease  is  a  severe  form  of  cachexia  accompanied  by  fever,  anaemia,  hypertrophy  of 
the  spleen,  wasting  of  the  muscles  and  diarrhrea.  It  used  also  to  be  described  as  tropical 
splenomegaly  but  is  now  known  to  be  the  same  disease  as  Kala  Azar. 


798 


THE  LEISHMANIOSES 


The  parasites  are  almost  always  intra-cellular,  generally  in  large  endothelial 
cells,  sometimes  in  leucocytes,  and  occasionally  in  the  red  cells  of  the  blood. 
A  large  number  of  parasites  are  often  found  in  one  cell  and  as  many  as  50  to 
200  have  been  found  within  a  rounded  amorphous  mass  of  tissue  representing 
probably  a  disintegrated  cell. 


Ti, 


FIG.  384. — Leishmania  donovani.  (After  Guiart.)  a,  Leishmania  in  a  macro - 
phage  ;  b,  c,  d,  parasites  in  the  spleen  ;  e,  longitudinal  division  ;  f,  multipart* - 
tion ;  g,  h,  i,  j,  cultivation  forms. 

Leishmania  donovani  occurs  as  a  small  rounded  or  oval  organism  measuring 
2-4/x  in  diameter.  When  stained  by  Romanowsky's  or  Leishman's  stain  it 
is  found  to  possess  a  large  spherical  poorly-staining  nucleus,  and  a  short 
rod-shaped  deeply-staining  blepharoplast  arranged  perpendicularly  or  tan- 
gentially  to  the  nucleus  ;  occasionally  a  prolongation  in  the  form  of  a  tail  i& 
seen,  attached  perpendicularly  to  the  blepharoplast ;  this  probably  repre- 
sents the  remains  of  a  flagellum  (fig.  384  d).  Reproduction  is  generally  by 

simple   longitudinal   division   (fig.  384  e), 
occasionally  by  multiple  fission — in  which 
case  the  mother-parasites  are  larger  and 
I  spherical    and    show    several    chromatin 

•»  masses  arranged  in  couples  (nucleus  and 

blepharoplast)  around  which  the  protoplasm 
segments  (fig.  384  f). 

't  [Sections. — For  the  recognition   of  the 

parasite  in  sections  of  tissues  the  follow- 
*f  ing  methods  are  recommended    (Nattan- 

i|  Larrier). 


•- 


FIG.  385. —Leishmania  donovani.  x  1000. 
Smear  from  spleen  from  a  case  of  Kala 
Azar  (Leishman's  stain). 


Fixation. — Any  of  the  following  fixatives 
are  good  provided*  the  pieces  of  tissue  be  small. 
(8X4X3  mm.) 

(i)  Acid  perchloride. 

(ii)  Saturated    aqueous    solution    of    per- 
chloride. 

(iii)  Alcohol.     Leave  the  tissue  in  70  per 
cent,  alcohol  for  3  hours,  then  in  80  per  cent,  for  3  hours  and  finally  in  90  per  cent, 

vl  malin*  The  tissue  may  be  Placed  in  2  per  cent,  formalin  for  3  hours 
and  then  passed  through  alcohol  as  above. 

Embed  in  paraffin. 

Staining. — The  best  results  are  obtained  with  the  following  procedures  : — 

(i)  Carbol-thionin.— Stain  for  30  min.  Wash  in  distilled  water.  Dehydrate 
rapidly  in  absolute  alcohol.  Differentiate  in  oil  of  cloves  and  afterwards  in  absolute 
alcohol.  Clear  in  xylol.  The  nuclei  are  shown  very  prominently. 

(11)  Kernschwarz  and  carbol-thionin.— Stain  in  Kernschwarz  for  15  min.  Wash 
well  in  distilled  water.  Stain  in  carbol-thionin  as  above  (i). 


INDIAN  KALA  AZAR 

(iii)  Alum-carmine  and  carbol-thionin. — Stain  in  the  carmine  solution  for  24 
hours.  Wash  for  30  mins.  Stain  for  30  min.  in  carbol-thionin.  Differentiate  in 
oil  of  cloves  until  the  section  has  a  red- violet  tint  and  the  protoplasm  of  the  cells 
a  pinkish  colour.  Pass  rapidly  through  alcohol  and  clear  in  xylol.] 

Appearance  in  cultures. — Rogers  found  that  the  parasites  multiplied  in 
blood  from  the  spleen  withdrawn  by  splenic  puncture  and  also  in  human 
blood  containing  a  small  amount  of  a  sterile  5  per  cent,  solution  of  sodium 
citrate  made  slightly  acid  with  citric  acid.  The  most  suitable  temperature 
is  22°  C.  though  multiplication  also  takes  place  at  27°  C.  Sub-cultures  do 
not  grow. 

In  acidulated  citrated  blood  growth  is  rapid  :  after  incubating  for  about 
48  hours  a  few  flagellated  forms  are  visible  ;  some  of  these  have  the  shape  of 
a  grain  of  barley  while  others  are  much  longer.  Each  parasite  has  a  nucleus 
and  a  blepharoplast,  the  latter  giving  origin  to  the  flagellum  :  the  anterior 
flagellated  extremity  is  rounded,  the  posterior  pointed  :  there  is  no  undulating 
membrane. 

Leishmania  donovani  can  also  be  grown  on  Novy  and  MacNeal's  medium. 

Novy  and  MacNeal's  medium  : 

Maceration  of  beef,       .....  1000  c.c. 


Agar, 

Peptone, 

Salt, 

Normal  solution  of  sodium  bicarbonate, 


20  grams. 
20      „ 
5       „ 
10  c.c. 


Dissolve  in  tubes,  sterilize  and  to  two  parts  of  the  alkaline  agar  add  1  part  defibrinated 
rabbit  blood. 

Nicolle's  medium  is  easier  to  prepare  and  gives  better  results. 
Nicolle's  medium— [N.N.N.  medium]  : 

Water,         -  -        900  c.c. 

Salt,  6  grams. 

Agar,  16 

Dissolve,  distribute  in  tubes,  sterilize  and  add  to  the  medium  in  each  tube  after 
liquefying  and  cooling  to  40°-50°  C.  one-third  its  volume  of  rabbit  blood  obtained  by 
cardiac  puncture.  Slope  the  tubes  for  twelve  hours,  incubate  at  37°  C.  for  five  days  to 
test  the  sterility  of  the  medium  and  then  keep  them  at  the  ordinary  temperature  of  the 
laboratory  for  a  few  days  before  sowing  them.  [The  tubes  should  be  sealed  to  prevent 
evaporation.] 

[Sub-cultures  can  be  obtained  on  this  medium  (Row).] 
[Laveran  and  Pettit's  medium : 

Peptone  (Chapoteaut),       -  2  grams. 

Salt,        -  6 

Water,  -  -         900  c.c. 

Dissolve.     Distribute  in  quantities  of  15  c.c.  in  small  Roux  flasks.     Sterilize. 

Add  to  each  flask  an  equal  volume  of  defibrinated  rabbit's  blood.     The  medium 

should  form  a  shallow  layer  on  the  bottom  of  the  vessel. 

[This  liquid  medium  is  useful  for  growing  rich  cultures  such  as  are  required 
for  animal  inoculation.  ] 

Etiology. — The  life  history  of  Leishmania  donovani  outside  the  human 
body  is  still  imperfectly  known.  It  may  be  conjectured  that  kala  azar  is 
propagated  by  a  biting  insect.  Patton  from  recent  experiments  is  inclined 
to  incriminate  the  bug,  Cimex  rotundatus  :  after  feeding  these  insects  on 
persons  suffering  from  the  disease  Patton  found  in  them  flagellated  forms 
similar  to  those  seen  in  cultures. 

[Patton  has  now  been  able  to  follow  the  complete  development  of  Leishmania 
donovani  in  both  the  European  and  Indian  bed  bugs  (Cimex  lectularius  and 
C.  rotundatus).  The  insects  were  infected  by  allowing  them  to  feed  upon  a  case 
of  kala  azar  in  whose  blood  the  parasites  were  present  in  large  numbers.  The 


800  THE  LEISHMANIOSES 

development,  as  in  the  case  of  Herpetomonas,  takes  place  in  three  stages — 
a  pre -flagellate,  a  flagellate  and  a  post-flagellate.  The  parasite  is  ingested  with 
the  white  cells  of  the  blood  and  passes  to  the  mid-gut  of  the  bug  where  it 
elongates  into  flagellated  forms  similar  to  those  seen  in  cultures  and  often 
forms  masses  of  rosettes.  About  the  seventh  to  the  ninth  day  after  a  single 
feed  the  mature  flagellated  parasites  undergo  a  further  developmental  process 
resulting  in  the  rounding  up  of  the  parasite  into  its  post-flagellate  form.  The 
latter  appears  on  about  the  eighth  day  and  the  changes  are  completed  by  the 
twelfth  day.  The  post-flagellate  forms  resemble  the  pre-flagellate  forms  in 
shape  only  and  differ  from  the  latter  in  being  slightly  larger  and  in  presenting 
very  different  staining  reactions — the  protoplasm  instead  of  staining  blue  as 
in  the  pre-flagellate  forms  stains  pink. 

[In  order  to  observe  these  changes  the  insects  must  have  only  one  feed.  If 
fed  during  the  flagellating  stage  the  parasites  are  destroyed.  In  several  of  the 
bugs  which  were  fed  once  only  Patton  observed  an  enormous  multiplication 
of  the  parasites  and  masses  of  rosettes  were  seen. 

[Patton  is  of  opinion  that  the  post-flagellate  forms  gain  entrance  to  a  new 
host  by  being  regurgitated  from  the  gut  while  the  bug  feeds,  for  the  whole  of 
the  multiplicative  stage  takes  place  in  the  mid-gut. 

[Donovan  has  failed  to  get  the  same  results  as  Patton  and  from  his 
observations  is  inclined  to  suspect  a  tick  (Conorrhinus  rubrofasciatus).] 

On  the  other  hand,  Manson  points  out  that  the  organisms  are  present  in 
small  numbers  in  the  peripheral  blood  while  they  abound  in  the  ulcers  on  the 
skin  and  intestinal  mucous  membrane  :  infection  by  contamination  with 
infected  excretal  matter  might  therefore  be  of  great  importance  in  the  pro- 
pagation of  the  disease  and  non-biting  flies  might  possibly  play  a  part. 

Experimental  inoculation. — [Until  recently]  all  attempts  to  inoculate  the 
disease  into  vertebrate  animals  have  failed. 

[Patton  inoculated  a  white  rat  intra-peritoneally  with  3  c.c.  of  an  emulsion 
of  the  spleen  from  a  case  of  Indian  kala  azar.  Fifteen  days  later  a  second 
inoculation  of  1  c.c.  was  given.  The  rat  died  two  hours  after  the  second  inocu- 
lation. Post  mortem  the  liver  and  spleen  were  considerably  hypertrophied  and 
contained  large  numbers  of  typical  parasites. 

[Laveran  inoculated  into  the  peritoneal  cavity  of  a  mouse  0'5  c.c.  of  an 
emulsion  of  bone  marrow  and  spleen  from  an  heavily  infected  dog  (L.  donovani). 
A  month  later  the  animal  was  killed.  Post  mortem  there  was  a  slight  peritoneal 
exudate  and  the  spleen  was  four  to  five  times  its  normal  size.  Numerous 
parasites  were  found  in  films  made  from  the  internal  organs.  ] 

2.  Leishmania  infantum. 

Cathoire  was  the  first  to  notice  a  peculiar  disease  in  Tunis  which  was 
clinically  like  kala  azar  but  only  affected  young  children  and  especially 
infants  1-2  years  old,  never  being  seen  in  children  more  than  6  years  of  age. 
Cathoire's  observations  were  confirmed  by  Nicolle  and  Cassuto.  A  similar 
disease  has  been  described  as  occurring  in  Italy — Infantile  splenic  anaemia 
(Pianese  and  others),  [and  in  Greece — Ponos]. 

This  disease  is  due  to  a  parasite  microscopically  identical  with  Leishmania 
donovani  but  from  which  it  differs  in  its  cultural  characteristics  and  in  the 
results  obtained  on  inoculation  into  animals.  Nicolle  has  provisionally  classed 
it  as  a  new  species  ;  Leishmania  infantum. 

[Leishman  gives  the  following  as  the  differences  between  Mediterranean  and 
Indian  kala  azar. 

["  1.  The  infantile  attacks  almost  exclusively  young  children,  while  the  Indian 
is  met  with  at  all  ages.  2.  Certain  differences  of  symptomatology  have  been 


MEDITERRANEAN  KALA  AZAR  801 

described.  3.  Cultures  of  the  parasites  are  readily  obtainable  upon  Novy  and 
MacNeal's  medium  in  the  case  of  L.  infantum,  and  are  easily  sub-cultivated,  while 
in  the  case  of  L.  donovani  cultures  on  this  medium  are  as  a  rule  unsuccessful  and 
sub-cultures  cannot  be  made.  On  the  other  hand  cultures  of  L.  donovani  succeed 
in  citrated  splenic  blood  and  usually  fail  in  the  case  of  L.  infantum.  4.  Inoculation 
of  the  spleen  parasites  into  dogs  and  monkeys  reproduces  the  disease  in  the  case 
of  L.  infantum  and  fails  in  L.  donovani.  5.  A  spontaneous  infection  of  dogs  has 
been  found  in  the  endemic  areas  of  infantile  kala  azar  but  no  such  infection  of  dogs 
has  been  encountered  in  India."] 

Detection  in  the  tissues. — The  parasite  is  invariably  found  in  the  material 
obtained  by  puncture  of  the  spleen  ;    the  organisms  are  less  numerous  in 
material  obtained  by  puncture  of  the  liver,  and  in  the  majority  of  cases  they 
cannot   be   found  in  the   peripheral   blood 
(Nicolle). 

Cultures. — The  organism  will  not  grow  in  ••    ^*         0 

Rogers'  medium  (citrated  blood),  but  cul-  ^    * 

tures  are  easily  obtained  in  Novy-MacNeaPs      -^~^  • 

or  in  Nicolle's  medium  (vide  ante).  . 

When    sown    in    Nicolle's    medium    and 

incubated  at  22°  C.  growth  begins  about  the  *        0* 

end  of  the  first  week  and  reaches  its  maxi- 
mum  on  the  twelfth  day.     The  organism  3jf      1 
will   be   found  alive  at   the   end   of  three  , 
months  and  sub-cultures  can  be  sown  in-                •        * 
definitely  (Nicolle). 

Inoculation  experiments -Dogs  can  be 
infected  by  inoculating  them  either  into 
the  peritoneal  cavity  or  into  the  liver  with  an  emulsion  of  an  infected 
spleen  :  the  distribution  of  the  parasite  is  the  same  as  in  man.  The  disease 
usually  runs  a  benign  course  though  several  animals  have  died  of  the  disease 
after  inoculation. 

In  monkeys  (Macacus  sinicus,  M.  cynomolgus),  inoculation  gives  rise  to  a 
disease  more  severe  than  in  dogs  and  running  a  more  rapid  course. 

When  mice  and  guinea-pigs  are  inoculated  into  the  peritoneal  cavity  with 
an  emulsion  of  tissue  from  dogs  containing  a  large  number  of  organisms,  the 
latter  multiply  in  the  peritoneal  cavity  but  do  not  become  generalized  and 
give  rise  to  no  symptoms  of  disease  (Laveran  and  Pettit).  [Nicolle,  Yakimoff 
and  Kohl-Yakimoff  obtained  a  generalized  infection  in  white  mice  by  the 
inoculation  of  emulsions  of  infected  tissues  or  of  cultures  into  the  vein  of  the 
tail.  Post  mortem  the  spleen  was  hypertrophied  and  parasites  were  found  in 
the  spleen  and  liver. 

[Dogs  can  be  infected  with  infected  dog  tissue  by  intra-peritoneal  inoculation 
(Senevet).] 

According  to  Nicolle  cultures  are  not  virulent ;  Novy  however  has  been 
able  to  infect  dogs  with  cultures  [and  vide  supra]. 

Etiology. — Nicolle  believes  that  infantile  kala  azar  is  of  canine  origin. 
[Gabbi,  and  Patton  however  do  not  consider  that  infantile  kala  azar  is  of 
canine  origin.  Patton  believes  that  the  bed  bug  Cimex  lectularius  will  be  found 
to  act  as  the  insect  porter  of  the  disease  seeing  that  L.  donovani  develops 
equally  in  C.  lectularius  and  in  C.  rotundatus.  ]  In  Tunis,  dogs  suffer  from  a 
natural  leishmaniosis  but  though  the  spontaneous  disease  is  more  benign  than 
the  inoculated  disease  Nicolle  believes  that  the  parasite  is  the  same  in  the  two 
cases.  In  several  cases  where  children  were  attacked  they  had  played  with 
dogs  which  were  obviously  ill. 

[Naturally  infected  dogs  have  been  found  also  in  Algiers  (Ed.  and  Et. 

3E 


802  THE  LEISHMANIOSES 

Sergent)  and  in  Greece  (Cardamatis).  The  percentage  infected  is  much  greater 
in  the  summer  months  than  in  winter.  A  cat  was  also  found  suffering  from  the 
spontaneous  disease  in  Algiers  (Ed.  and  Et.  Sergent,  Lombard  and  Quilichini).  ] 

3.  Leishmania  tropica. 

Syn. — Leishmania  furonculosa. — Helcosoma  tropicum. 

The  endemic  granulomata  of  warm  countries  (Oriental  sore,  Aleppo  boil, 
Delhi  boil,  and  known  also  by  innumerable  other  local  names)  are  caused  by 
a  protozoan  organism,  discovered  by  Wright  (Helcosoma  tropicum)  and  by 
Marzinowsky  and  Bogrow,  and  belonging  to  the  genus  Leishmania. 

Appearance  in  the  tissues. — In  the  tissues  the  parasite  is  indistinguishable 
from  the  parasite  of  kala  azar  :  it  occurs  within  the  cells,  generally  in  the 
macrophages,  endothelial  cells  and  mononuclear  leucocytes,  but  occasionally 
in  the  polymorphonuclear  leucocytes  and  in  cells  of  the  connective  tissue. 

Appearance  in  culture. — In  cultures  L.  tropica  is  distinguished  from  L. 
donovani  by  the  early  division  of  the  flagellum  (some  cells  have  two  flagella 
at  their  anterior  end)  and  by  the  greater  length  and  more  marked  flexuosity 
of  the  flagellum  (Nicolle  and  Sicre). 

[Kow  found  the  following  differences  between  L.  donovani  and  L.  tropica 
when  the  two  parasites  were  grown  on  Nicolle 's  medium  at  the  same  time. 

[1.  The  growth  of  L.  tropica  is  much  more  luxuriant  than  that  of  L.  donovani 
over  the  same  period.  2.  L.  tropica  is  distinctly  larger  than  L.  donovani. 
3.  The  posterior  extremity  of  L.  donovani  is  distinctly  more  pointed  than  that 
of  any  other.  4.  There  is  a  far  larger  number  of  fine  vacuoles  in  the  fully 
formed  flagellates  of  L.  donovani  than  in  L.  tropica.  5.  L.  tropica  is  charac- 
terized by  the  appearance  of  fine  chromatin  particles  distributed  in  the  body  of 
the  parasites  just  as  L.  donovani  is  hollowed  out  by  the  presence  of  fine 
vacuoles.  These  last  two  however  may  be  purely  accidental.] 

Experimental  inoculation. — The  parasite  of  Oriental  sore  can  be  inoculated 
into  man  and  also  into  monkeys  (Macacus  sinicus)  (Marzinowsky). 

[^Etiology. — Leishmania  tropica  forms  flagellates  in  bugs  (Wenyon,  Patton). 
Patton  has  no  doubt  but  that  in  Cambay  the  bug  Cimex  rotundatus  is  the  only 
transmitter  of  the  disease  in  spite  of  the  fact  that  his  attempts  to  prove  the 
hypothesis  experimentally  have  failed.  Wenyon  states  that  in  Bagdad  bugs 
are  not  sufficiently  numerous  to  account  for  the  prevalence  of  the  disease  there 
and  concludes  that  the  house  fly  is  sometimes  the  transmitting  agent  but  more 
usually  one  of  the  mosquitoes  or  the  sand  fly  Phlebotomus.  ] 

[The  disease  encountered  in  South  America  and  known  as  Espundia  is  due 
to  a  parasite  closely  resembling  L  tropica  but  as  there  are  certain  differences 
between  the  two  organisms  Laveran  and  Nattan-Larrier  prefer  at  present  to 
regard  the  South  American  parasite  as  a  variety  of  the  latter— L.  tropica  var. 
americana.  ] 

Other  species  of  Leishmania. 

Three  other  species  of  Leishmania  have  been  described  as  occurring  in  man 
but  the  existence  of  these  parasites  and  their  identity  are  still  matters  of 
doubt.  One  of  them  was  described  in  Typhus  fever  by  Lewaschew  and  by 
Gotschlich  [but  see  p.  847]  :  the  second,  in  dengue  by  Graham  :  and  the 
third  by  Wilson  and  Chowning  in  the  spotted  fever  of  the  Eocky  Mountains, 
a  disease  transmitted  by  a  tick  (Dermacentor  occidentalis)  ;  Stiles  and 
Kicketts  however  have  failed  to  confirm  this  last  discovery. 


CHAPTER  LXII. 
THE  FLAGELLATA. 

Section  I. — The  Trypanosomata. 
Introduction. 

1.  Trypanosoma  lewisi,  the  rat  trypanosome,  p.  805. 

Trypanosomes  in  rodents  other  than  rats,  p.  808. 

2.  Trypanosoma  equiperdum,  the  trypanosome  of  Dourine,  p.  809. 

3.  Trypanosoma  brucei,  the  trypanosome  of  Nagana,  p.  811. 

African  Trypanosomiases  related  to  Nagana,  p.  813. 

4.  Trypanosoma  evansi,  the  trypanosome  of  Surra,  p.  814. 

5.  Trypanosoma  equinum,  the  trypanosome  of  Mai  de  Caderas,  p.  814. 

6.  Trypanosoma  theileri,  the  trypanosome  of  Galziekte,  p.  816. 

7.  The  trypanosomes  of  Sleeping  sickness,  p.  816. 

Trypanosoma  gambiense,  p.  816. 
Trypanosoma  rhodesiense,  p.  820. 

8.  Trypanosoma  cruzi,  p.  822. 

9.  Trypanosomes  in  birds,  p.  823. 

10.  Trypanosomes  in  cold-blooded  vertebrata,  p.  824. 
Section  II. — Trichomonas  vaginalis,  p.  825. 

Other  species  of  Trichomonas,  p.  826. 
Section  III. — Lamblia  intestinalis,  p.  827. 

THE  Flagellata  are  free  Protozoa  characterized  by  the  presence  of  one  or 
more  flagella  (which  are  totally  different  structures  from  bacterial  flagella) 
and  sometimes  by  an  undulating  membrane.  Numerous  parasites  of  man 
and  the  lower  animals  belong  to  this  group. 


SECTION  I.— THE  TRYPANOSOMATA. 

The  Trypanosomata  live  as  parasites  in  the  blood  of  man  and  a  large 
number  of  the  lower  animals.  They  are  flagellated  organisms  with  fusiform 
bodies,  a  centrally-situated  nucleus,  and  a  laterally  placed  undulating  mem- 
brane. The  free  thickened  border  of  this  membrane  terminates  behind  in 
the  posterior  half  of  the  body  in  a  centrosome  (kineto-nucleus)  or  blepharo- 
plast,  while  in  front  it  is  as  a  rule  prolonged  into  a  free  flagellum.  A  large 
vacuole  is  often  visible  towards  the  posterior  part  of  the  body  and  in  the 
same  part  of  the  parasite  chromatin  granules  staining  deeply  with  nuclear 
dyes  are  also  found. 

In  the  blood  reproduction  takes  place  by  longitudinal  binary  fission  (schizo- 
gony)  :  the  newly  formed  elements  may  undergo  further  division  before  they 
separate  and  by  a  repetition  of  this  process  a  rosette  arrangement  is  produced 
(vide  infra). 


804 


THE   FLAGELLATA 


The  parasites  are  [in  the  majority  of  cases]  transmitted  by  the  bites  of 
blood-sucking  insects  in  which  the  Trypanosomes  multiply  by  sexual  repro- 
duction (sporogony)  and  of  which  the  details  are  still  imperfectly  known 
[but  see  T.  equiperdum  and  T.  equinum.] 


FIG.  387. — The  Trypanosomidce.    (After  Guiart.)      A,  Trypanusumu  , 
C,  Herpetomonas  ;    D,  Spirochceta  ;  E,  Treponema. 


B,  Trypanoplasma  ; 


Schaudinn  distinguished  several  genera  of  the  Trypanosomidse. 

1.  The  genus  Trypanosoma :   having  an  undulating  membrane  and  one  flagellum. 

2.  The  genus  Trypanoplasma :    having  in  addition  to  an  undulating  membrane 
and  an  anterior  flagellum,  a  second  free  flagellum  inserted  into  the  posterior  part 
of  the  body. 

3.  The  genus  Herpetomonas  :  having  a  free  flagellum  but  no  undulating  membrane 
(p.  805). 

4.  The  genus  Spirochaeta :    elongated,  sinuous  organisms  having  an  undulating 
membrane,  but  no  flagellum. 

5.  The  genus  Treponema :  elongated,  sinuous  organisms  having  a  short  flagellum 
at  each  end  but  no  undulatory  membrane. 

It  has  already  been  pointed  out  that  this  classification  is  not  universally  accepted 
and  that  by  some  observers  the  spirochsetes  and  treponemes  are  grouped  with  the 
bacteria  (p.  711). 

Methods  of  examination.— In  searching  for  trypanosomes  fresh  blood 
should  be  examined  in  the  same  way  as  for  the  haematozoa  (p.  771).  For 
prolonged  observation,  such  as  the  study  of  agglutination,  hanging  drop 
preparations  (p.  132)  luted  with  vaseline  or  paraffin  are  necessary.  To 
prevent  coagulation  the  blood  should  be  mixed  with  citrated  normal  saline 
solution  : — 

Water,        -         -  -       1000  grams. 

Sodium  citrate,    -  6       „ 

Sodium  chloride,  6       „ 

The  citrated  blood  may  also  be  defibrinated.  Or  the  blood  may  be  allowed 
to  coagulate  ;  the  trypanosomes  then  pass  into  the  serum  and  can  be  studied 
unhampered  by  the  red  cells  (Francis). 

For  stained  preparations,  the  best  method  is  that  of  Laveran,  using  BorreFs 
blue  (p.  772).  In  the  case  of  most  of  the  Trypanosomes  found  in  the  mam- 
malia it  is  sufficient  to  stain  for  5  or  10  minutes,  but  in  the  case  of  Trypano- 
soma lewisi  the  staining  must  be  continued  for  20  minutes  (p.  806  fig.  388 
appearances  produced  by  staining). 

Any  of  the  methods  described  as  useful  for  the  Hcematozoa  are  applicable 
to  the  staining  of  the  Trypanosomata.  One  of  the  best  stains  is  Laveran's 


THE  RAT  TRYPANOSOME  805 

modification  of  Giemsa's  stain  (p.  774)  :  it  is  specially  useful  for  staining  the 
trypanosomes  of  Nagana  and  Mai  de  Caderas. 

Levaditi  advises  drying  the  blood  films  in  the  air  and  then  fixing  them  in 
alcohol-ether.  After  fixing,  the  films  are  stained  first  in  a  saturated  aqueous 
solution  of  Bismarck-brown  and  washed,  and  then  stained  for  2  minutes  in 
Unna's  polychrome  blue  diluted  with  an  equal  volume  of  water,  washed, 
dried  and  mounted  in  balsam. 

When  the  number  of  trypanosomes  in  the  blood  is  small,  thick  films  should 
be  spread  on  slides  and  the  haemoglobin  dissolved  out  either  by  Ross'  or  Le 
Dantec's  method  (p.  772).  Laveran  recommends  fixing  in  absolute  alcohol 
and  dissolving  the  hsemoglobin  in  a  1  per  cent,  solution  of  acetic  acid.  Dutton 
and  Todd  aspirate  a  drop  of  blood  and  a  drop  of  citrated  normal  saline  solu- 
tion into  a  capillary  tube  with  a  bulb  ;  the  fluids  are  mixed  and  the  tube 
sealed  in  the  flame  and  centrifuged  ;  the  red  cells  collect  in  the  capillary  part 
of  the  tube,  and  above  them  at  the  lower  part  of  the  bulb  is  a  layer  of  leuco- 
cytes with  which  the  parasites  are  mixed  and  this  is  used  for  examination. 

For  the  study  of  the  cytology  of  trypanosomes  the  following  is  the  only  method 
which,  according  to  Salvin  Moore  and  Breinl,  gives  satisfactory  results. 

Fixation. — Coat  a  slide  with  a  thin  layer  of  glycerin- albumin  and  spread  a  drop 
of  blood  over  it,  before  drying  dip  the  slide  into  a  strong  solution  of  Flemming's 
solution  for  5—10  minutes,  wash  in  alcohol  of  progressively  increasing  strength  up  to 
absolute  alcohol  then  treat  with  iodine  and  iodide  in  80  per  cent,  alcohol  and  finally 
in  30  per  cent,  alcohol. 

Staining. — Stain  with  Heidenhain's  iron-hsematoxylin  containing  a  few  drops  of 
a  solution  of  lithium  carbonate. 

Cultivation. — Attempts  have  been  made  to  cultivate  Trypanosomes  outside 
the  body  and  reference  will  be  made  to  the  results  later  in  the  chapter.  The 
medium  used  is  blood-agar  :  either  Novy  and  MacNeaPs  or  Nicolle's  may  be 
employed  (p.  799).  Mathis  recommends  ordinary  nutrient  agar  to  which, 
after  liquefying  and  cooling  to  59°  C.,  1-2  parts  of  defibrinated  blood 
(rabbit,  rat,  guinea-pig,  ox  or  horse)  are  added  :  the  mixture  is  heated  to 
75°-800  C.  for  half  an  hour,  then  solidified  on  the  slope.  Whatever  the 
medium  there  must  be  a  certain  amount  of  water  of  condensation  at  the 
bottom  of  the  tubes  and  into  this  the  infected  blood  is  sown.  India-rubber 
caps  should  be  slipped  over  the  mouths  of  the  tubes  to  prevent  the  medium 
drying  up. 

1.  Trypanosoma  lewisi.1 
The  rat  trypanosome. 

This  parasite  was  discovered  by  Gros  and  Chaussat  in  the  field  mouse,  mole 
and  black  rat,  and  has  been  found  by  Lewis,  Crookshank,  Danilewsky,  Laveran 
and  Mesnil  and  others  in  Mus  decumanus,  M.  rattus,  M.  refuscens,  Cricetus 
frumentarius,  etc.  It  occurs  in  a  large  percentage  of  rats  all  over  the  world. 

The  parasite  is  transmitted  by  the  rat  flea,  [Ceratophyllus  fasciatus,  and  by 
other  fleas]  and  by  the  rat  louse,  Hcematopinus  spinulosus  (Prowazeck). 

[The  normal  method  of  transmission  is  that  the  ripe,  infective  form  of  the 
Trypanosome — the  final  form  of  the  developmental  cycle  which  it  passes 
through  in  the  flea — -is  regurgitated  from  the  stomach  of  the  flea  into  the 
wound  made  by  the  proboscis  of  the  flea  during  the  act  of  feeding.  Rats 
can  be  infected  by  devouring  infected  fleas  but  this  is  not  the  usual  method 
by  which  the  transmission  of  the  Trypanosome  from  rat  to  rat  is  effected  by 

1  The  trypanosome  of  the  rat  is  often  described  as  Herpetomonas  lewisi  (Kent).  This 
description  is  inaccurate  because  the  fundamental  characteristic  of  the  genus  Herpeto- 
monas (type  H.  muscce  domesticce)  is  the  absence  of  an  undulating  membrane  (vide 
ante}. 


806 


THE   FLAGELLATA 


the    flea ;    on   the   contrary,    it   is   an    exceptional    and    aberrant    method 

(Minchin  and  Thomson).] 
Appearance  in  the  blood. — In  fresh  blood,  T.  lewisi  is  flattened  and  fusiform 

and  often  twisted  on  itself.  It  is  the  most  motile  of  all  the  Trypanosomes 
and  vigorously  displaces  the  red  cells  :  it  may  sometimes 
be  seen  moving  across  the  preparation  like  a  dart  with  the 
flagellum  in  front.  Its  length  including  the  flagellum  is 
24-25/x,  its  breadth  1'5/x  (Laveran  and  Mesnil).  Its  protoplasm 
is  finely  granular;  the  [tropho-]  nucleus  is  not  visible  but  the 
centrosome  [kineto-nucleus]  appears  as  a  refractile  spot  to- 
wards the  posterior  end. 

In  preparations  fixed  and  stained  by  Laveran's  method 
(p.  772)  the  protoplasm  is  stained  pale  blue  with  fine  granules  ; 
the  [tropho-]  nucleus,  oval  in  shape  and  situated  in  the 
anterior  one-third  of  the  body,  is  stained  lilac.  The  undu- 
lating membrane  is  unstained — with  the  exception  of  its  free 
thickened  border  which  is  stained  lilac — and  is  continued 
anteriorly  into  the  flagellum,  while  posteriorly  it  takes  origin 
from  the  centrosome  which  is  stained  deep  violet. 

In  the  blood  of  rats  which  have  been  infected  for  a  long 
time  only  fully  developed  fusiform  trypanosomes  are  seen,  all 
of  the  same  length. 

To  study  the  multiplication  forms  it  is  necessary  to  prepare 
stained  films  from  the  blood  of  a  rat  which  has  been  inocu- 
lated intra-peritoneally  4—8  days  previously. 

The  peritoneal  exudate,  which  contains  numerous  multiplication 
forms  during  the  first  2  or  3  days,  is  not  a  very  suitable  material 
for  the  study  of  the  cytology  of  the  parasites. 

Before  dividing,  the  Trypanosome  increases  in  size,  the 
centrosome  approaches  the  nucleus  and 
the  flagellum  thickens  at  the  end  which 
is  in  relation  to  the  centrosome.  Soon  the 
nucleus  and  centrosome  as  well  as  the 
base  of  the  flagellum  divide.  The  newly 
formed  flagellum  separates  from  the 
original  one  and  though  at  first  much 
shorter  than  the  latter,  it  rapidly  elongates 
while  the  protoplasm  is  dividing.  The 
young  Trypanosome  finally  separates  but 
may  again  subdivide  before  detaching 
itself. 

Other  multiplication  forms  have  the 
appearance  of  spherical  or  oval  bodies  in 
which  the  nuclei  and  centrosome  having 
approached  each  other  divide  (into  from 
2  to  16  parts),  and  the  flagella  at  the  same 
time  split  into  two  without  the  protoplasm  dividing :  then  the  protoplasm 
shows  a  series  of  notches  around  its  periphery  (rosette  appearance)  and 
finally  divides  into  as  many  parts  as  there  are  nuclei.  The  small  parasites 
resulting  from  the  segmentation  of  the  rosette  may  again  in  their  turn 
divide. 

Appearance  in  cultures. — Novy  and  MacNeal  were  able  to  grow  the  organism 
on  their  blood-agar  medium  (p.  799)  :  growth  takes  place  best  at  the  tem- 
perature of  the  laboratory  and  is  poorer  at  34°-37°  C.  These  observers  have 


FIG.  388.— Try- 
panosoma  lewisi 
stained  by  Laver- 
an's  method. 
(After  Laveran 
and  Mesnil.) 
x  about  2000  dia- 
meters. 


^ 


FIG.   389. — Trypanosoma   leurisi. 
Leishman's  stain. 


1000. 


THE  RAT  TRYPANOSOME 


807 


been  able  to  sub-cultivate  the  organisms  for  22  generations  ;  the  cultures  are 
pathogenic  and  contain  living,  motile  Trypanosomes.  In  old  cultures  rosette 
forms  appear  ;  these  become  more  and  more  numerous  and  the  culture  dies 
after  about  15-20  days  in  the  warm  incubator,  but  at  a  much  later  period  if 
kept  at  the  temperature  of  the  laboratory. 


FIG.  390. — Multiplication  forms  of  Trypanosoma  lewisi.     (After  Laveran  and  Mesnil.)     1,  adult 
trypanosome  ;  2,  a  trypanosome  about  to  divide  ;  3,  rosette  forms  ;  4,  young  forms. 


The  size  of  the  Trypanosomes  varies  considerably  in  the  same  culture.  Some  may 
be  50-60/z  long  while  others  do  not  exceed  1-2/x  in  length  including  the  flagellum. 
This  fact  explains  why  it  has  been  found  possible  to  infect  rats  with  the  filtrates 
of  cultures  passed  through  a  Berkefeld  bougie. 

The  shape  also  of  the  Trypanosomes  is  very  varied,  and  pyriform,  rounded,  and 
delicate  fusiform  parasites  are  seen.  Numerous  agglomerated  masses  occur  in 
which  the  flagella  are  always  directed  towards  the  centre. 

Agglomeration  (Agglutination).- — Laveran  and  Mesnil  have  shown  that 
Trypanosoma  lewisi  will  live  much  longer  in  blood  kept  in  the  ice  chest 
(5°-7°  C.)  than  at  the  temperature  of  the 
laboratory.  In  infected  rat  blood  mixed 
with  an  equal  volume  of  saline  solution 
and  defibrinated  Trypanosomes  will  retain 
their  vitality  for  30-50  days  if  kept  in  the 
cold,  whereas  if  kept  at  a  temperature  of 
15°-20°  C.  they  will  die  in  about  3  days. 

In  blood  kept  in  this  way,  Trypanosomes 
at  first  preserve  their  normal  appearance 
and  are  very  motile.  But  after  about  3 
days  agglomeration  commences  :  two  Try- 
panosomes unite  by  their  aflagellar  ex- 
tremities, others  join  them  and  form  a  sort 
of  rosette,  the  flagellated  ends  of  all  remain- 
ing free  and  motile ;  the  number  of  parasites 
entering  the  agglomerated  mass  increases 
daily.  The  blood  is  pathogenic  so  long  as 
it  contains  motile  parasites.  Agglomeration 
occurs  much  more  rapidly  in  hanging  drops  of  the  blood  kept  at  laboratory 
temperature :  under  these  conditions  agglomerated  masses  may  be  observed 
after  24  hours. 


FIG.  391.— Agglomeration  of 
Trypanosoma  lewisi. 


808  THE  FLAGELLATA 

On  adding  some  immunized  rat  serum  or  the  normal  serum  of  certain  other 
animals  (especially  fowl,  horse,  dog,  sheep,  or  rabbit)  to  defibrinated  blood 
or  serum  containing  Trypanosomes  agglomeration  takes  place  in  a  few 
minutes  and  is  often  complete.  The  rosettes  may  then  collect  together 
forming  enormous  masses  visible  to  the  naked  eye.  The  parasites  constituting 
these  masses  are  motile.  Disagglomeration  is  sometimes  observed  (Laveran 
and  Mesnil) :  the  trypanosomes  which  were  instantaneously  agglomerated 
by  the  serum  again  become  free  in  a  few  hours.  While  the  property  of 
agglomeration  appears  rapidly  in  the  blood  of  immunized  animals,  the 
paralyzing  property  (leading  to  the  immobilization  of  Trypanosomes  whether 
agglomerated  or  not)  is  only  seen  in  hyperimmunized  animals. 

Experimental  inoculation. — Rats  are  readily  infected  with  Trypanosoma 
leivisi,  but  with  the  exception  of  the  guinea-pig — in  which  a  very  short  and 
abortive  infection  occurs — no  other  animals  are  susceptible.  Infected  blood 
or  a  culture  will  reproduce  the  disease  when  inoculated  into  the  peritoneal 
cavity,  into  the  blood  stream,  sub-cutaneously  or  even  into  the  stomach  of  the 
rat.  Rats  become  infected  by  consuming  the  blood  of  infected  rats  (Francis  : 
not  confirmed  by  Laveran), 

The  most  certain  method  of  infection  is  by  intra-peritoneal  inoculation.  During 
the  first  3  days  following  the  inoculation  the  parasites  multiply  in  the  peritoneal 
cavity  and  numerous  reproduction  forms  can  be  seen.  Then  the  trypanosomes 
disappear  from  the  peritoneal  cavity,  but  appear  in  the  blood  where  they  rapidly 
multiply :  multiplication  forms  are  however  less  numerous  in  the  blood  than  they 
are  in  the  peritoneal  cavity.  At  the  end  of  a  week  the  trypanosomes  cease  to 
multiply  in  the  blood,  and  for  a  period  varying  from  20  days  to  4  months  only 
slender  adult  parasites  are  seen  (latent  period). 

The  number  of  parasites  in  the  blood  varies.  Sometimes  there  are  as  many  as 
1  to  every  2  or  3  red  cells. 

Generally  speaking,  the  presence  of  Trypanosomes  in  the  blood  of  the  rat 
is  unaccompanied  by  any  symptoms  of  disease  ;  in  some  cases,  however,  the 
infection  may  prove  fatal  (Jiirgens,  Francis,  and  others). 

Immunity. — Rats  in  which  parasites  appeared  in  the  blood  after  a  first 
inoculation  of  trypanosomes  never  show  a  blood  infection  on  subsequent 
inoculation.  The  serum  of  rats  which  have  been  inoculated  several  times 
with  trypanosome  blood  is  prophylactic,  and  if  inoculated  at  the  same  time 
as  the  parasites  into  the  peritoneal  cavity  of  a  normal  rat  the  trypanosomes 
do  not  pass  into  the  blood  (Kempner  and  Rabino witch)  :  it  fails  however 
to  cause  the  elimination  of  trypanosomes  from  the  blood  of  infected  rats 
during  the  latent  period  (Laveran  and  Mesnil). 

The  serum  of  immunized  rats  has  powerful  agglutinating  properties  and 
rapidly  leads  to  rosette  formation  when  diluted  from  five  to  fifty  times. 

Trypanosomes  in  rodents  other  than  rats. 

Jolyet  and  de  Nabias  have  found  trypanosomes  in  the  blood  of  four  out  of  ten 
rabbits  examined  in  Bordeaux.  The  trypanosome-infected  rabbits  were  generally 
thin  and  wasted,  and  suffered  from  diarrhoea.  The  trypanosomes  were  long,  and 
including  the  flagellum  measured  30-36/x ;  the  body  was  nucleated,  cylindrical  in 
the  centre,  pointed  behind  and  terminated  anteriorly  in  a  flagellum  :  the  undulating 
membrane  was  very  narrow  and  could  only  be  seen  after  staining.  In  fresh  blood 
the  parasites  were  highly  motile. 

Trypanosomes  have  also  been  found  in  rabbits  by  other  observers  (M.  Nicolle, 
Petrie),  as  well  as  in  guinea-pigs  (Ktinstler),  mice  (Dutton  and  Todd),  squirrels 
(Donovan,  Laveran),  ground-squirrels  (Chalachnikow),  etc. 


THE   TRYPANOSOME   OF  DOURINE  809 

2.  Trypanosoma  equiperdum  (Doflein). 
Trypanosome  of  Dourine.1 
8301. — Trypanosoma  rougeti. 

The  trypanosome  of  dourine  was  discovered  in  Constantinople  by  Rouget, 
in  the  blood  of  a  stallion  affected  with  mal  du  coit.  It  has  been  studied  more 
recently  by  Schneider  and  Buffard. 

Horse  syphilis  is  transmitted  by  the  act  of  coitus,  perhaps  also  by  fleas  (Rabino- 
witch  and  Kempner,  Sieber  and  Gonder).  In  the  first  stage  of  the  disease  in  the 
stallion  there  is  oedema  of  the  penis,  scrotum  and  inguinal  regions  ;  in  the  mare  this 
painless  oedema  affects  the  vulva  and  vagina  and  leads  to  a  more  or  less  abundant 
mucous  secretion.  In  the  second  stage — about  a  month  after  coitus — characteristic 
infiltrated  firm  plaques  appear,  affecting  the  sub-cutaneous  tissue  covering  the  ribs, 
the  crupper  and  sometimes  the  neck,  shoulders  and  thighs.  Finally,  in  the  third 
stage,  extreme  anaemia,  paralysis  and  sometimes  epileptiform  attacks  appear. 
Recovery  is  exceptional  and  when  it  does  occur  is  as  a  rule  merely  apparent,  the 
animal  soon  suffering  a  relapse. 

Morphology.— The  trypanosome  of  dour- 
ine is  fusiform  in  shape  and  measures  about 
25-28/A  long  by  2/x  broad  in  the  centre.  Its 
protoplasm  stains  uniformly  blue  by  La- 
veran's  method  and  contains  no  chromatin 
granules.  The  nucleus  is  distinctly  cen- 
trally situated.  There  is  a  folded  un- 
dulating membrane  the  free  border  of 
which  terminates  anteriorly  in  the  flagellum 
and  is  lost  posteriorly  in  the  centrosome. 
It  is  distinctly  less  motile  than  T.  lewisi 
but  nevertheless  shows  obvious  movements 
of  translation :  the  motility  is  still  apparent 
after  about  18  hours  in  preparations  of 
fresh  blood. 

Reproduction  appears  to  take  place  in  FlG- 
the  same  way  as  in  T.  lewisi ;  binary  longi- 
tudinal fission  is  the  method  most  frequently  seen.  Forms  with  8  to  10 
nuclei  developing  into  a  sort  of  rosette  have  been  recorded  (Laveran,  Rabino- 
witch,  Kempner). 

Cultures. — All  attempts  to  grow  Trypanosoma  equiperdum  outside  the  body 
in  the  blood  of  susceptible  animals  have  failed  (Rouget) :  in  such  blood  the 
parasite  loses  its  virulence  in  less  than  24  hours. 

Experimental  inoculation. — Cold-blooded  animals,  birds,  cattle,  monkeys 
and  guinea-pigs  are  immune. 

Mice,  white  rats,  rabbits,  dogs,  horses  and  mules  are  all  susceptible  to 
infection. 

In  determining  the  susceptibility  of  a  given  animal  to  the  parasite  it  is  necessary 
to  take  into  account  the  adaptation  which  the  parasite  undergoes  as  a  result  of 
repeated  passages  through  a  given  species  (Nocard).  Rouget  experimenting  with 
rats  and  mice  recovered  a  parasite  which  proved  to  be  very  virulent  for  these  animals. 
Schneider  and  Buffard  after  several  passages  through  dogs  recovered  a  trypanosome 
which  was  harmless  for  mice  and  rats  ;  this  trypanosome  was  inoculated  by  Nocard 
into  the  brain  of  a  young  rat,  which  it  infected,  and  it  was  then  found  to  be  virulent 
for  adult  rats. 

1  [Dourine,  Horse  syphilis,  or  Mal  du  coit  is  a  disease  of  horses  occurring  in  Europe, 
North  America,  Algeria  and  India.  ] 


810  THE   FLAGELLATA 

Infection  of  susceptible  animals  is  very  easily  effected  by  inoculating  a 
trace  of  blood-stained  serous  exudate  or  a  few  cubic  centimetres  of  infected 
blood  sub-cutaneously,  intra-peritoneally  or  intra-venously  :  it  is  sufficient 
even  to  place  a  drop  of  the  blood-stained  exudate  on  a  superficial  excoriation 
of  the  skin  or  on  an  uninjured  mucous  membrane  to  infect  the  animal. 
Attempts  at  infection  by  ingestion  have  always  failed.  In  one  instance 
the  seminal  fluid  of  a  rabbit  contained  the  parasite  and  the  animal  infected 
an  healthy  doe  by  the  genital  passage  (Rouget). 

Mice.  White  rats. — In  these  animals  the  parasite  rapidly  becomes  general- 
ized :  mice  die  in  5-6  days,  the  blood  and  internal  organs  swarming  with 
trypanosomes.  Infection  cannot  however  be  obtained  in  every  case  and  the 
results  vary  with  trypanosomes  from  different  sources  :  many  strains  fail 
altogether  to  produce  an  infection. 

Rabbits. — In  infected  rabbits  the  trypanosomes  are  found  only  inter- 
mittently in  the  blood.  The  animals  suffer  from  an  irregular  fever,  but 
there  is  no  relation  between  the  paroxysms  of  fever  and  the  occurrence  of 
trypanosomes  in  the  blood.  The  rabbits  exhibit  certain  characteristic 
symptoms;  for  instance,  oedema  and  sloughing  of  the  ear,  muco-purulent 
conjunctivitis,  swelling  and  sloughing  of  the  external  genitalia,  paraplegia 
and  cachexia.  Death  takes  place  after  2-4  months. 

Horses. — About  the  fourth  day  following  the  sub-cutaneous  inoculation 
of  the  parasite,  there  is  an  oedematous  infiltration  of  the  cellular  tissue  about 
the  site  of  inoculation  ;  the  exudate  contains  numerous  leucocytes  and  some 
feebly  motile  trypanosomes  which  however  are  larger  than  the  parasites 
found  in  films  of  the  blood.  About  the  sixth  day  the  nuclei  of  the  trypano- 
somes are  seen  to  be  divided  into  two  or  three  masses  :  then  the  oedema 
increases  rapidly  and  forms  a  tumour  containing  a  blood-stained  exudate 
in  which  the  parasites  are  present  in  considerable  number. 

According  to  Schneider  and  Buflfard,  the  trypanosomes  found  in  the  exudate  are 
of  various  shapes. 

1.  Adult  trypanosomes  similar  to  those  just  described. 

2.  Large,  pyriform,  non-motile  bodies  with  appendices  not  unlike  the  posterior 

segments  of  the  trypanosomes :  this  V-shaped  form — which,  seen  from  above, 
is  like  a  comb  or  a  squid — represents  longitudinal  division  of  the  parasite 
and  is  similar  to  the  figures  seen  in  Trypanosoma  lewisi. 

3.  Trypanosomes  arranged  in  pairs  or  groups  of  four  radiating  from  a  central 

point  like  a  star  and  formed  by  the  meeting  of  their  posterior  ends  :  this 
represents  a  later  stage  of  longitudinal  fission. 

The  parasites  in  the  exudate  begin  to  diminish  in  number  from  the  eighth 
to  the  tenth  day  and,  together  with  the  oedematous  tumour,  soon  disappear. 
The  developmental  cycle  of  the  trypanosome  lasts  about  a  week. 

After  this  the  parasites  are  present  only  in  small  numbers  in  the  peripheral 
blood,  but  occur  in  large  numbers  in  the  plaques  which  now  soon  make  their 
appearance. 

Schneider  and  Buffard  think  that  the  plaques  seen  in  dourine  are  due  to  a  secondary 
multiplication  of  the  trypanosomes  in  the  capillaries  of  the  skin — where  they  are 
arrested — and  that  the  young  forms  resulting  from  this  division  reinfect  the  blood 
stream  :  "  the  hsemorrhagic  foci  and  areas  of  softening  found  in  the  central  nervous 
system  are  also  produced  by  the  migration  of  the  trypanosome  into  the  medullary 
vessels  which  it  blocks  and  perforates." 

Asses. — Asses  are  less  susceptible  to  the  parasite  of  dourine  than  horses  : 
the  succession  of  forms  is  less  regular,  and  the  swelling  assumes  considerable 
proportions  from  the  first ;  when  the  oedema  subsides,  the  parasite  passes 
into  the  general  circulation.  The  trypanosomes  which  are  present  at  first 
in  large  numbers  in  the  blood  soon  become  fewer  and  fewer  :  then,  6-8  days 


THE   TRYPANOSOME   OF  NAGANA 


811 


after  the  first  swelling  has  disappeared,  a  second  swelling  occurs  at  the  site 
of  inoculation,  in  which  the  parasites  multiply  and  soon  cause  a  fresh  infection 
of  the  blood.  The  disease  runs  a  distinctly  intermittent  course  and  the 
parasite  only  multiplies  at  the  site  of  inoculation. 

Dogs. — Dogs  in  Europe  are  highly  susceptible.  The  initial  swelling  lasts 
a  long  time  and  the  trypanosome  only  infects  the  blood  stream  after  an 
interval  of  15  or  16  days.  It  is  probable  that  subsequently  multiplication 
takes  place  in  all  the  organs  of  the  body.  The  most  pronounced  symptoms 
are  conjunctivitis,  keratitis,  and  hypopyon,  oedema  of  the  external  genitalia, 
and  paralyses. 

Indian  dogs  are  highly  immune  to  the  dourine  of  India  (Pease,  Lingard). 

Immunity. — Rouget  has  shown  that  the  blood  of  infected  rabbits  and  dogs 
collected  in  the  last  stages  of  the  disease  has  immunizing  properties.  Such 
blood,  injected  as  a  prophylactic  either  alone  or  mixed  with  the  virus,  protects 
mice  against  infection.  It  has  no  therapeutic  properties. 

3.  Trypanosoma  brucei. 

The  trypanosome  of  Nagana. 

Sir  David  Bruce  has  shown  that  Nagana  or  "  Tsetse-fly  disease  "  (Glossina 
morsitans)  is  due  to  a  trypanosome. 

Nagana  is  a  disease  affecting  horses,  asses,  mules,  oxen,  dogs  etc.  in  South 
East  and  Central  Africa. 

The  disease  is  transmitted  by  the  Tse-tse  fly  (Glossina  morsitans).  [It  can  also  be 
conveyed  from  infected  to  non-infected  animals  under  experimental  conditions  by 
Glossina  palpalis].  It  is  probable  that  the  fly  often  infects  itself  by  biting  wild 
animals  (buffaloes,  etc.)  whose  blood  not  infrequently  contains  trypanosomes 
although  the  animals  themselves  show  no  signs  of  disease  (Bruce). 

Carnivora  (dogs,  etc.)  appear  to  contract  the  disease  by  feeding  upon  the  flesh 
of  animals  dead  of  Nagana.  It  seems  also  to  be  proved  that  animals  infected  with 
the  disease  may  transmit  the  infection  by  biting  healthy  animals  :  such  transmission 
may  possibly  in  the  cases  observed  have  been  due  to  the  presence  in  the  saliva  of 
infected  blood  from  erosions  of  the  gums. 

Morphology. — Trypanosoma  brucei  is  found  in  varying  numbers  in  the 
blood  of  infected  animals. 

In  the  fresh  condition  the  parasite  occurs  as  a  motile  vermicule  having  an 
undulating  membrane  and  an  anterior  flagellum  :  the  posterior  end  is  some- 
times filiform,  sometimes  rounded  or  like 
a  section  of  a  fir  cone.  The  movements 
though  very  active  are  prolonged,  and 
darting  movements  like  those  seen  in  T. 
leivisi  are  never  observed.  All  the  para- 
sites are  much  of  the  same  size  ;  some 
are  broader  than  others  and  have  two 
undulating  membranes  (a  stage  of  multi- 
plication). 

In  fixed  and  stained  preparations,  the 
parasites  measure,  including  the  flagel- 
lum, 26-27/n  in  length  and  1'5-2'5/x  in 
breadth.  In  horses'  and  asses'  blood 

the      length    generally    reaches    2S-33/*.     left,  an  adult  trypanosome ;    on  the  right,  a 

The  protoplasm  is  stained  rather  deeply    Ssp^e0sst0a^s)undergoing  division  (three 
by  Laveran's  method  and  has  a  number 

of  large,  deeply-staining  granules  in  its  anterior  part.  The  nucleus  is 
centrally  situated,  oval  in  shape,  and  stains  rather  less  deeply  than  the 


FIG.   393. — Trypanosome   of   Nagana    (Tr. 
brucei).     (After  Laveran  and  Mesnil.)     On  the 


812  THE   FLAGELLATA 

centrosome    in    which    the    flagellum    terminates    [cf.    morphology    of    T. 
gambiense,  p.  818]. 

Multiplication  takes  place  by  binary  longitudinal  fission  (Laveran  and 
Mesnil) ;  this  commences  with  division  of  the  centrosome,  and  is  soon  followed 
by  division  of  the  flagellum,  nucleus  and  protoplasm  (fig.  393). 

Involution  forms. — Under  unfavourable  conditions  involution  forms  make  their 
appearance  :  the  trypanosomes  become  stumpy,  roll  up  into  balls  and  form  small 
agglomerations.  These  forms  were  taken  by  Bradford  and  Pliinmer  to  represent 
stages  of  multiplication. 

When  a  trypanosome  dies  the  cytoplasm  disappears  first,  then  the  nucleus,  and 
finally  only  the  flagellum  and  centrosome  remain. 

Cultures. — Novy  and  MacNeal  obtained  cultures  of  Tr.  brucei  on  their 
blood-agar  medium.  Cultures  often  remain  sterile  so  that  it  is  advisable 
to  sow  a  large  number  of  tubes  to  ensure  a  successful  result.  Growth  takes 
place  between  25°  and  34°  C.,  but  the  higher  the  temperature  the  more 
quickly  do  the  parasites  die  :  at  the  temperature  of  the  laboratory  a  culture 
may  remain  alive  for  45  days.  Novy  and  MacNeal  have  succeeded  in  sub- 
cultivating  to  the  fourteenth  generation. 

Cultures  grown  at  25°  C.  are  seldom  as  virulent  as  trypanosomes  from 
the  blood  and  only  kill  rats  and  mice  in  7-10  days  instead  of  in  3-5  :  at 
34°  C.  they  rapidly  lose  their  virulence. 

In  cultures  the  trypanosomes  are  generally  arranged  either  in  pairs  con- 
nected by  their  aflagellar  ends,  or  in  rosette-shaped  colonies  consisting  of 
10  to  20  individuals,  the  flagella  appearing  to  be  situated  at  the  periphery 
of  the  rosette. 

Agglomeration. — Outside  the  body  the  trypanosomes  in  the  blood  of 
infected  animals  soon  exhibit — apart  from  the  involution  forms  which  have 
been  described — the  phenomena  of  agglomeration  (Laveran  and  Mesnil, 
Martini).  Forms  similar  to  those  which  have  already  been  described 
as  occurring  in  cultures — association  of  two  parasites  and  rosette-forma- 
tion— are  seen.  The  agglomerated  masses  may  break  up  after  a  variable 
time. 

Agglomeration  is  hastened  by  the  addition  of  serum  from  a  dog,  horse,  sheep,  pig, 
monkey,  etc.  Human  serum  exhibits  no  agglomerating  action,  and  the  serum  of 
an  immunized  goat  has  no  more  agglomerating  action  than  a  normal  goat's  serum. 
The  serum  of  a  cow,  immunized  by  Nocard,  was  highly  agglomerating  but  had  no 
trypanicidal  properties. 

Experimental  inoculation. — Trypanosoma  brucei  will  readily  infect  most  of 
the  mammalia.  Man  however  is  [thought  to  be]  immune. 

Inoculation  is  followed  by  infection  whether  the  blood  of  the  infected  animal 
(which  is  the  material  generally  used)  be  inoculated  sub-cutaneously,  intra- 
venously, or  intra-peritoneally. 

Infected  blood,  if  kept  free  from  contamination,  loses  its  virulence  outside  the 
body  in  a  few  days  whether  it  be  kept  in  the  ice  chest  or  at  laboratory  temperature 
(Laveran).  Desiccation  also  renders  it  harmless. 

The  incubation  period  varies  for  a  given  species  both  with  the  number  of 
parasites  inoculated  and  with  the  condition  of  the  parasites.  The  disease  is 
always  acute  and  fatal  in  mice,  rats,  dogs  and  monkeys ;  subacute  in  rabbits, 
guinea-pigs,  horses,  asses  and  pigs ;  and  chronic  in  cattle,  sheep  and  goats : 
these  last  may  recover  from  infection.  Grothusen  and  Martini  have  suc- 
ceeded in  infecting  a  zebra  :  this  fact  is  in  contradiction  to  the  immunity 
which  the  zebra  seems  to  possess  in  nature. 

The  most  constant  symptoms  in  all  animals  are  oedema,  fever,  anaemia, 
wasting  and  paralyses.  In  rats  and  mice  the  parasites  multiply  until  at 


THE  TRYPANOSOME  OF  NAGANA         813 

the  time  of  the  death  of  the  animal  they  number  as  many  as  the  red  cells : 
in  other  animals  they  are  not  so  numerous. 

Immunity. — Laveran  has  shown  that  human  serum  has  a  specific  action 
on  infected  animals.  When  human  serum  (1-2  c.c.  for  a  rat  weighing  200 
grams)  is  inoculated  sub-cutaneously  into  an  infected  rat  or  mouse,  the 
parasites  rapidly  disappear  from  the  blood  :  but  they  reappear  after  4r-S 
days.  By  repeating  the  injections  the  life  of  the  infected  animals  can  be 
considerably  prolonged,  though  a  permanent  cure  is  never  obtained. 

Human  serum  is  feebly  prophylactic.  Thus,  when  inoculated  at  the  same 
time  as  the  trypanosomes  it  will  sometimes  prevent  infection  but  the  animal 
is  not  immunized  :  if  inoculated  24  hours  before  the  virus  infection  is 
delayed.  The  serum  of  all  the  lower  animals  (monkeys,  etc.)  is  without  any 
action. 

Some  species  of  animals  (cattle,  goats  and  sheep  for  example)  often  recover 
from  an  attack  of  Nagana,  and  they  are  then  immune  to  the  disease  and 
their  blood  has  prophylactic  properties  against  Tr.  brucei  (Laveran). 

Schilling  and  Koch  have  devised  a  means  of  vaccinating  cattle.  The  method 
consists  in  using  a  trypanosome  which  has  been  passed  two  or  three  times  through 
dogs  or  through  dogs  and  rats  alternately :  the  passage  virus  sets  up  usually  a  mild 
infection  in  cattle  and  the  animals  become  immune,  but  occasionally  a  severe  infec- 
tion is  produced.  To  avoid  this  Schilling  increased  the  number  of  passages,  first 
passing  the  trypanosome  seven  times  through  dogs  and  rats  alternately,  then 
eighteen  to  twenty  times  through  dogs  only.  It  is  stated  that  bovine  animals  resist 
inoculation  with  this  passage  virus  very  well  and  that  their  serum  exhibits  bac- 
tericidal properties  for  the  trypanosome :  but  the  efficiency  of  the  method  has  yet 
to  be  confirmed.  It  is  inapplicable  in  the  case  of  horses  since  these  animals 
succumb  to  the  inoculation  of  the  passage  virus. 

Martini  succeeded  in  keeping  alive  for  a  long  time  two  asses  which  had  resisted 
inoculation  with  a  trypanosome  after  it  had  been  passed  through  mice  and  which 
had  been  subsequently  inoculated  five  times  intra-venously.  The  serum  of  these 
asses,  which  ultimately  died,  proved  to  be  prophylactic  for  mice.  At  the  time  of 
death  their  blood  was  infective  for  the  dog. 

Laveran  and  Mesnil  have  tried,  but  unsuccessfully,  to  produce  immunity 
by  inoculating  animals  with  an  attenuated  virus  (attenuation  being  effected 
by  age,  heat,  cold,  mixing  with  toluidine  blue,  etc.).  The  incubation  period 
following  the  inoculation  of  the  attenuated  trypanosomes  is  longer  than 
normal,  but  once  the  disease  develops  it  runs  its  ordinary  course. 

African  Trypanosomiases  related  to  Nagana. 

Animals  in  Africa  are  subject  to  many  trypanosome  infections.  The  parasites  in 
these  diseases  are  all  more  or  less  like  T.  brucei  and  may  be  divided  into  several 
species. 

Trypanosoma  soudanense. — Szewczyk,  and  Rennes  have  observed  an  epizootic 
among  horses  in  the  French  Sudan  (Mai  de  la  Zousfana)  running  a  chronic  course 
and  having  as  its  causal  agent  a  trypanosome  (Trypanosoma  soudanense)  similar  to 
that  of  Surra  (Laveran). 

The  same  parasite  is  responsible  for  the  disease  of  dromedaries  observed  by 
Cazalbou  in  the  French  Sudan  and  known  as  Mbori  or  "  the  fly  disease,"  and  of 
the  disease  of  dromedaries  described  by  Ed.  and  Et.  Sergent  in  Algeria  and  known 
as  El  Debab.1 

Trypanosoma  dimorphon,  studied  by  Button  and  Todd  in  Gambia,  which  infects 
mules,  cattle,  sheep  and  horses  in  French  Guinea,  Dahomey,  the  Congo  and  Senegal 
is  also  closely  related  to  the  trypanosome  of  Nagana. 

Trypanosoma  congolense  (Brodden)  has  been  found  in  the  Congo  in  sheep  and 
asses  in  the  neighbourhood  of  Leopoldville,  and  in  cattle,  dogs  and  sheep  near 
Brazzaville.  It  is  very  closely  related  to  T.  dimorphon. 

1  [El  Debab,  the  fly  or  horse  fly.] 


814  THE   FLAGELLATA 

Trypanosoma  cazalboui  (Laveran)  is  the  cause  of  a  disease  of  horses  and  cattle 
in  the  Soudan  known  as  Souma. 

Trypanosoma  pecaudi  (Laveran)  is  the  infecting  agent  in  Bal£ri,  a  disease  of 
horses  and  other  beasts  in  the  Soudan. 

4.  Trypanosoma  evansi. 

The  trypanosome  of  Surra. 

Evans  described  a  disease,  Surra,1  affecting  horses,  elephants  and  camels  in 
India,  which  is  due  to  a  trypanosome  other  than  that  which  causes  Nagana. 

[But  since  it  has  been  shown  that  Mbori,  a  disease  of  dromedaries  in  the  Sudan, 
is  caused  by  a  species  of  trypanosome  similar  to  that  of  Surra  this  latter  disease  can 
now  no  longer  be  regarded  as  limited  to  India,  and  Laveran  is  of  the  opinion  that 
other  African  epizootic  diseases  due  to  trypanosomes  other  than  T.  brucei  may  also 
be  varieties  of  Surra.  ] 

T.  evansi  on  inoculation  will  produce  an  infection  in  rats,  mice,  dogs, 
monkeys,  cattle,  horses,  asses  and  mules. 

Infected  horses  and  mules  invariably  die  of  an  acute  disease,  but  bovine 
animals  and  sheep  and  goats  often  recover  after  suffering  from  a  sub-acute 
or  chronic  infection.  Animals  which  recover  are  immune  to  the  disease. 
In  Bos  indicus  (the  Indian  sacred  bull)  the  immunity  does  not  appear  to  last 
more  than  2  years  (Wryburg). 

Morphologically,  T.  evansi  and  T.  brucei  are  identical,  though  perhaps  the 
former  is  more  slender  and  more  motile  than  the  latter  (p.  811)  and  contains 
fewer  chromatin  granules. 

The  two  parasites  are  however  specifically  different,  and  the  diseases  to 
which  they  give  rise  cannot  be  regarded  as  identical  (Laveran  and  Mesnil). 
Goats  hyper-immunized  against  Nagana  are  as  susceptible  to  Surra  as  non- 
immunized  goats  (Laveran  and  Mesnil),  and  a  Brittany  cow  which  had 
recovered  from  Nagana  and  was  hyper-immunized  against  that  disease  proved 
as  susceptible  to  Surra  as  did  a  normal  animal  of  the  same  race  (Nocard). 

It  is  difficult  to  obtain  cultures.  Laveran  and  Mesnil  only  succeeded  once 
in  six  experiments  :  the  organism  died  out  after  the  first  sub-culture. 

The  disease  appears  to  be  transmitted  by  a  fly  of  the  genus  Tabanus  and 
perhaps  also  by  a  Stomoxys.  [Possibly  by  more  than  one  species  of  each  of 
these  genera.  In  any  case  Surra  is,  like  Nagana,  mainly  propagated  by 
biting  flies  (Laveran  and  Mesnil).] 

5.  Trypanosoma  equinum. 

The  trypanosome  of  Mai  de  Caderas. 

Mai  de  Caderas  2  is  a  fatal  disease  of  Equidae  in  South  America  :  it  is  charac- 
terized by  fever,  progressive  wasting,  profound  anaemia  and  paralysis  of  the 
hind  quarters.  The  disease  is  contagious,  but  the  channels  of  infection  are 
absolutely  unknown,  so  that  it  is  not  even  agreed  whether  biting  insects 
play  any  part  in  the  spread  of  the  disease  or  not. 

[Sexual  intercourse  does  not  give  rise  to  infection  as  it  does  in  the  case  of  dourine 
(Lignieres).  The  only  fact  upon  which  all  observers  are  agreed  is  that  the  Capybara 
(Hydrochcerus  capybara)  is  the  source  from  which  the  carrier  of  the  disease  probably 
obtains  its  supply  of  the  virus.  When  the  farmers  in  Paraguay  find  dead  Capy- 
baras  on  their  farms,  they  know  that  caderas  will  soon  break  out  among  the  horses. 
There  is  a  striking  analogy  between  this  mortality  among  the  Capybaras  which 

1  [Surra  is  the  word  which  has  been  used  from  time  immemorial  by  the  natives  of  certain 
parts  of  India  to  denote  a  disease  of  horses,  characterized  by  profound  cachexia  without 
any  lesion  being  found  post  mortem  to  account  for  it  (Laveran  and  Mesnil).  ] 

2  [Mai  de  caderas  denotes  disease  of  the  hind-quarters  and  is  so  called  from  the  paralysis 
of  that  part  of  the  body  which  is  so  characteristic  a  symptom  of  the  disease.  ] 


THE   PARASITE   OF  MAL  DE  CADERAS  815 

precedes  epizootics  of  Mai  de  caderas  and  that  among  rats  which  precedes  epidemics 
of  plague.  Lignieres  has  infected  rats  with  Mai  de  caderas  by  inoculating  them 
with  fleas  crushed  in  salt  solution  (Nabarro's  translation  of  Laveran  and  Mesnil's 
"  Trypanosomes  ").] 

Morphology  of  the  parasite. — Trypanosoma  equinum  was  discovered  by 
Elmassian  [in  Paraguay  in  1901].  In  fresh  preparations  it  is  morphologically 
indistinguishable  from  T.  brucei  and  T.  evansi  ;  in  stained  preparations  how- 
ever the  characters  of  the  centrosome  differentiate  it  clearly  from  allied 
parasites. 

The  centrosome  is  very  small,  and  since  it  stains  like  the  flagellum  it  is 
difficult  to  distinguish.  [In  trypanosomes  of  the  type  T.  evansi,  on  the  other 
hand,  the  centrosome  is  very  obvious  and  measures  about  0'5/>t  and  stains 
deep  purple  (Laveran  and  Mesnil).] 

T.  equinum  measures  22-24/x  long  by  about  1'5/*  broad.  It  multiplies 
like  T.  brucei  by  binary  longitudinal  fission. 

Agglomeration  in  the  blood,  which  is  favoured  by  the  addition  of  normal 
sheep,  pig  or  horse  serum  and  particularly  of  serum  from  bovines,  sheep  or 
pigs  infected  with  caderas,  gives  rise  to  rosette  forms  in  which  the  posterior 
ends  are  in  apposition. 

The  number  of  trypanosomes  in  the  blood  of  infected  Equidse  varies  at  different 
periods  of  the  disease.  In  the  early  stages  the  parasites  are  very  scanty  but  as 
death  approaches  they  become  more  numerous  ;  they  are  not  however  constantly 
present  in  the  blood  in  the  course  of  the  naturally  contracted  disease,  and  there  are 
times  when  no  parasites  can  be  found.  Trypanosomes  are  found  when  the  tem- 
perature of  the  animal  rises  above  38°  C.  but  disappear  when  it  reaches  41°  C. 
(Elmassian  and  Migone). 

Vitality. — In  blood  kept  at  the  ordinary  temperature  Trypanosoma  equinum 
dies  rapidly  but  if  the  blood  be  kept  in  the  ice  chest  the  parasite  will  live  for 
3  days.  The  addition  of  normal  serum  (fowl,  horse,  sheep,  rat  or  bovine)  to 
the  blood  lengthens  the  life  of  the  parasite  by  from  5-11  days. 

[Cultures.— Laveran  and  Mesnil  failed  to  grow  T.  equinum  on  blood-agar 
at  room  temperature.  Thomas  and  Breinl  using  a  modification  of  Novy  and 
M'NeaPs  medium— chicken-broth-rabbit-blood  agar— grew  a  T.  equinum 
from  rabbits'  blood  at  22°  C.  and  infected  a  rat  after  29  days'  incubation  : 
sub-cultures  failed.] 

Experimental  inoculation. — Horses,  mules,  asses,  monkeys,  mice,  white 
rats,  guinea-pigs,  rabbits  and  dogs  are  all  susceptible  to  experimental  infec- 
tion. No  symptoms  are  produced  in  sheep,  cattle  and  pigs,  but  the  blood  of 
these  animals  remains  infective  for  mice  for  about  2  months  after  inoculation. 
Birds  are  immune. 

Human  serum  has  a  specific  action  on  animals  infected  with  Caderas 
(Laveran  and  Mesnil).  Laveran  was  able  to  cure  10  per  cent,  of  mice  experi- 
mentally infected  by  inoculating  them  with  human  serum. 

Sheep,  goats,  bovines  and  pigs  which  have  recovered  from  an  infection 
with  T.  equinum  are  immune  and  their  serum  has  for  a  short  time  some 
slight  prophylactic  property. 

Specific  nature  of  Mai  de  Caderas. — Caderas  can  be  differentiated  from 
other  Trypanosome  infections  by  a  study  of  the  immunity  reactions.  Dogs 
which  have  recovered  from  an  infection  with  the  parasite  of  dourine  and 
which  are  therefore  immune  to  that  disease  are  as  susceptible  to  Caderas  as 
normal  dogs  (Lignieres).  A  goat  and  a  sheep  cured  of  and  immune  to  Nagana 
were  just  as  susceptible  to  Caderas  as  normal  control  animals.  The  serum 
of  animals  cured  of  Nagana  has  no  action  on  T.  equinum  (Laveran  and 
Mesnil). 


816  THE  FLAGELLATA 

6.  Trypanosoma  theileri. 

Trypanosome  of  "  Galziekte." 

Cattle  in  the  Transvaal  [and  in  the  Orange  River  Colony,  Cape  Colony  and 
possibly  in  other  parts  of  South  Africa]  are  subject  to  a  disease,  known  to 
the  farmers  of  South  Africa  as  Galziekte  [gall-sickness],  which  is  characterized 
by  anaemia  and  may  or  may  not  be  accompanied  by  fever.  It  may  assume 
a  malignant  form  and  rapidly  terminate  in  the  death  of  the  animal.  Theiler 
found  a  trypanosome  in  the  blood  of  the  affected  animals  and  this  was 
described  [almost  simultaneously]  by  Laveran  [and  Bruce].  The  trypano- 
some of  Galziekte  ( T.  theileri)  is  the  largest  of  the  mammalian  trypanosomes, 
being  almost  twice  the  size  of  T.  brucei :  it  measures  60-70/x  long  by  3-4/w. 
broad  in  the  larger  forms,  while  the  smallest  forms  measure  from  25-30/x 
long  by  2-3/A  broad.  The  protoplasm  stains  deeply  and  is  very  granular  : 
the  nucleus  is  oval  and  centrally  situated  :  the  centrosome  is  rounded,  stains 
well  and  is  placed  some  little  distance  from  the  posterior  end  of  the  parasite. 
Multiplication  takes  place  by  binary  longitudinal  fission. 

Cattle  are  the  only  animals  susceptible  to  inoculation  with  T.  theileri. 
Cattle  which  survive  an  attack  of  the  disease  are  immune. 

The  disease  is  transmitted  by  the  bites  of  flies  of  the  family  Hippoboscidce 
(H.  rufipes  and  H.  macitlata).  [H.  maculata  is  very  rare  in  South  Africa 
where  it  appears  to  have  been  introduced  at  the  time  of  the  Boer  War  with 
cavalry  horses  coming  from  India  (Laveran).]  Theiler  has  succeeded  in 
infecting  healthy  animals  by  placing  on  them  Hippoboscce  taken  from  affected 
animals. 

7.  The  trypanosomes  of  sleeping  sickness. 

[Human  Trypanosomiasis.     Negro  Lethargy.     Trypanosome  fever.] 

A.  Trypanosoma  gambiense. 

In  1898  Nepveu  found  trypanosomes  in  the  blood  of  a  man  in  Algeria,  but 
his  descriptions  were  so  lacking  in  precision  as  to  make  his  conclusions 
doubtful. 

In  1902  Dutton  [in  conjunction  with  Forde]  discovered  trypanosomes  in 
the  blood  of  an  European  who  had  lived  in  Gambia  for  6  years  and  who  was 
suffering  from  a  disease  which  ultimately  proved  fatal.  The  disease  was 
characterized  chiefly  by  an  irregular  fever  which  did  not  yield  to  quinine, 
oedema  of  the  face  and  lower  limbs,  cutaneous  erythema,  hypertrophy  of 
the  spleen,  enlargement  of  the  lymphatic  glands  and  general  weakness. 
Dutton  described  the  parasite  as  Trypanosoma  gambiense. 

Manson  and  Daniels,  and  Manson  and  Broden  soon  afterwards  confirmed 
Dutton's  discovery  by  finding  trypanosomes  in  man  on  the  Congo,  and  cases 
of  human  trypanosome  fever  were  soon  afterwards  recorded  by  Baker  in 
Uganda,  Brumpt  in  the  Congo,  and  Forde  in  Uganda  ;  and  Dutton  and  Todd 
several  times  found  trypanosomes  in  natives  of  Gambia  suffering  from  mild 
indefinite  maladies. 

In  1903  Castellani  found  trypanosomes  in  the  cerebro-spinal  fluid  of  70 
per  cent,  of  persons  affected  with  sleeping  sickness.  The  observations  were 
soon  confirmed  by  Bruce  and  Nabarro  and  by  Brumpt.  Castellani's  trypano- 
some (Trypanosoma  ugandense]  is  practically  always  found  in  the  cerebro- 
spinal  fluid  [as  well  as  in  the  blood  and  gland  juice  (Nabarro)]  of  persons 
suffering  from  sleeping  sickness,  and  has  never  been  found  in  the  cerebro- 
spinal  fluid  of  persons  free  from  the  disease. 

Sleeping  sickness  is  the  cause  of  a  very  considerable  mortality  among  the  negroes 
of  the  West  Coast  and  centre  of  Africa :  for  some  years  past  it  has  decimated  the 
negro  population  of  Uganda  and  the  Great  Lakes.  The  disease  has  a  great  pre- 


THE  TRYPANOSOMES   OF  SLEEPING  SICKNESS        817 

dilection  for  the  negro  race  but  also  attacks  mulattos  and  several  cases  have  been 
recorded  in  Europeans. 

Further  observation  tended  to  show  that  the  Trypanosomes  of  Dutton 
and  of  Castellani  must  be  regarded  as  one  and  the  same  organism,  Trypano- 
soma  gambiense  (Bruce,  Nabarro  and  Greig,  Laveran).  Morphologically 
they  are  indistinguishable  :  and  the  difference  between  sleeping  sickness  and 
trypanosome  fever  is  merely  a  function  of  the  localization  of  the  same  trypano- 
some  in  the  body.  Sleeping  sickness  has  been  produced  in  the  monkey  by 
the  inoculation  of  Dutton's  trypanosome.  If  the  trypanosome  is  in  the 
blood,  the  disease  produced  is  Dutton's  disease,  trypanosome  fever ;  if  in  the 
cerebro-spinal  fluid  then  the  symptoms  are  those  of  sleeping  sickness.  [It 
must  be  noted  however  that  Nabarro  states  that  if  the  trypanosome  be 
carefully  looked  for  in  sleeping  sickness  patients  it  can  be  found  not  only  in 
the  cerebro-spinal  fluid  but  also  in  the  blood  and  gland  juices.]  Manson  has 
recorded  a  case  in  which  sleeping  sickness  developed  in  an  European  who  up 
till  that  time  had  only  shown  symptoms  attributed  to  Dutton's  trypanosome. 
Monkeys  which  have  acquired  an  immunity  against  T.  gambiense  are  also 
immune  against  T.  ugandense  [and  vice  versa;  but  subsequent  investigation 
has  shown  that  this  acquired  immunity  is  apparent  rather  than  real  and  the 
most  recent  work  tends  to  show  that  no  immunity  is  attainable  with  the 
trypanosome  of  sleeping  sickness  (Laveran,  Nabarro  and  others).  However 
it  is  now  almost,  if  not  quite,  universally  believed  that  T.  gambiense  and 
T.  ugandense  are  the  same  species  (Nabarro)]. 

[More  recent  investigations  have  however  thrown  some  doubt  upon  the 
identity  of  all  trypanosomes  found  in  man. 

[Stephens  and  Fantham  for  instance  have  isolated  a  trypanosome  from 
an  European  who  became  infected  in  some  part  of  North-East  Rhodesia. 
This  trypanosome  they  regard  as  a  new  species  (Trypanosoma  rhodesiense) 
and  in  this  opinion  they  are  supported  by  Laveran  from  his  own  experiments 
and  observations  (see  p.  820). 

[Castellani  also  is  disposed  to  revert  to  the  opinion  he  held  in  1903  that 
there  may  be  more  than  one  species  of  human  trypanosome,  possibly  trans- 
mitted by  the  same  fly,  in  the  same  way  that  different  species  of  malarial 
parasites  are  transmitted  by  the  same  mosquito.] 

Methods  of  detection. — As  a  general  rule  the  blood  and  cerebro-spinal 
fluid  should  be  examined  for  trypanosomes  and  as  the  latter  are  often  present 
only  in  very  small  numbers  the  fluid  should  be  centrifuged. 

Cerebro-spinal  fluid. — 10-15  c.c.  of  fluid  should  be  withdrawn  by  lumbar  puncture 
(p.  199).  The  first  few  drops  are  rejected  as  they  may  contain  a  little  blood  and 
this  would  interfere  with  the  examination.  The  fluid  should  then  be  centrifuged  at 
once  for  a  quarter  of  an  hour  and  the  whitish  deposit  used  for  making  films,  which 
must  be  stained  by  one  of  the  usual  methods  (p.  804).  [It  is  better  to  examine 
fresh  unstained  films  as  the  trypanosomes  obtained  by  centrifuging  cerebro-spinal 
fluid  do  not  stain  well.] 

Blood. — When  direct  examination  of  blood  films  has  failed  to  reveal  the  presence 
of  trypanosomes,  Bruce  and  Nabarro  collect  10  c.c.  of  blood  in  a  tube  containing  a 
little  [1  per  cent.]  potassium  citrate,  solution,  centrifuge  for  10  minutes,  pipette  off 
the  supernatant  plasma  [together  with  some  of  the  middle  layer  and  a  little  of  the 
red  corpuscle  layer]  and  centrifuge  this  again.  They  repeat  the  operation  four 
times  and  use  the  deposit  from  the  fourth  centrifugation  for  making  films  for 
microscopical  examination. 

The  method  of  Le  Dantec  (p.  772)  may  also  be  employed. 

To  complete  the  identification  of  the  organism  it  will  also  be  well  to  inoculate 
a  little  of  the  suspected  blood  or  cerebro-spinal  fluid  into  a  susceptible  animal 
(for  example,  into  the  peritoneum  of  a  rat). 

3F 


818 


THE  FLAGELLATA 


For  stained  preparations  the  deposit  obtained  on  centrifuging  the  cerebro- 
spinal  fluid  is  not  very  satisfactory  and  the  preparations  made  from  it  are 
always  poor.  Trypanosomes  stain  better  in  blood  films  but  as  they  occur 
only  in  small  numbers  in  human  blood  it  is  better  to  inoculate  an  animal 
and  stain  films  of  the  animal's  blood. 

Greig  and  Gray,  Dutton  and  Todd,  and  Beck  recommend  puncturing  an 
hypertrophied  lymphatic  gland  with  an  ordinary  hypodermic  syringe  and 
examining  the  drop  of  gland  juice  thus  obtained.  ["  Gland  puncture  is  by 
far  the  most  efficient  method  of  demonstrating  the  presence  of  trypanosomes 
in  cases  of  trypanosomiasis  "  (Dutton  and  Todd).] 

Nattan-Larrier  and  Tanon  have  always  been  able  to  find  the  parasite  in 
films  made  with  blood  obtained  by  scarification  of  the  erythematous  patches 
on  the  skin. 

Morphology. — The  human  trypanosome  has  all  the  ordinary  generic  char- 
acters of  other  trypanosomes.  It  is  highly  motile  [but  exhibits  but  little 
or  no  translatory  power  in  the  field  of  the  microscope  (Bruce)].  The  cyto- 
plasm contains  chromatin  granules  :  the  nucleus  is  oval  and  situated  in  the 
centre  of  the  parasite  :  the  undulating  membrane  is  narrow  :  and  the 
flagellum,  representing  as  a  rule  about  one-quarter  of  the  total  length  of  the 
trypanosome,  occasionally  has  no  free  portion  at  all,  the  cytoplasm  extending 
to  the  distal  extremity  of  the  flagellum.  The  centrosome  stains  well.  Some- 
times a  vacuole  is  seen  round  or  near  the  centrosome  :  [Laveran  and  Mesnil 
regard  this  as  a  result  of  deficient  technique,  but  Nabarro  thinks  that  vacuoles 
are  normally  present  at  times]. 

[The  trypanosome  is  15-33/*  long  and  in  breadth  averages  1'5/*  in  the  long 
to  2*5/*  in  the  short  and  stumpy  forms  (Bruce).  Great  differences  are 
sometimes  found  in  the  average  length  in  the  same  individual.  For  instance 
in  one  case,  an  European,  at  the  beginning  of  the  illness  the  trypanosomes 
averaged  17/x  in  length  whereas  at  a  later  date  they  averaged  25*8/*.]  Multi- 
plication takes  place  by  longitudinal  fission  (p.  803). 


FIG.  394. — Different    appearances    presented    by    Trypanosoma    gambiense. 
n,  Nucleus  ;  c,  Centrosome  ;  m',  m,  Undulating  membrane  ;  n,  flagellum. 

[T.  gambiense,  "  like  T.  brucei,  is  markedly  dimorphic.  In  size  and  general 
appearance  also  these  two  species  so  closely  resemble  one  another  that  one 
might  easily  believe  them  to  be  varieties  of  the  same  species.  There  are, 
however,  some  slight  differences  in  morphology,  .  .  .  but  whether  these  differ- 
ences will  bear  the  test  of  more  extended  observations  remains  to  be  seen  " 
(Bruce).] 


THE   TRYPANOSOMES   OF  SLEEPING  SICKNESS        819 


FIG.  395. — Trypanosoma gambiense.  For- 
mation of  the  latent  stage  and  transforma- 
tion of  the  latent  stage  into  a  trypanosome. 
(After  Guiart.) 


In  the  blood,  trypanosomes  are  occasionally  seen  in  pairs  with  their  pos- 
terior [aflagellar]  ends  crossed  (Laveran  and  Mesnil). 

When  the  trypanosomes  become  very  numerous  in  the  blood  they  pass  to 
the  spleen  and  bone  marrow,  and  subsequently  disappear  from  the  blood  of 
the  peripheral  circulation.  The  parasite 
assumes  a  new  character  in  the  spleen  :  a 
deep  band  makes  its  appearance  between 
the  centrosome  and  the  nucleus  and  the 
latter  becomes  surrounded  by  a  vacuole, 
the  trypanosome  disintegrates  and  is  re- 
duced to  a  nucleus  ;  this  represents  the 
latent  form  of  the  parasite.  The  nucleus 
soon  divides,  giving  rise  to  a  new  centro- 
some from  which  a  flagellum  takes  origin, 
thus  a  small  trypanosome  of  the  ordinary 
appearance  is  produced  (fig.  395)  ;  the 
latent  forms  disappear,  and  the  newly 
formed  parasites  pass  again  into  the  blood 
of  the  peripheral  circulation. 

Vitality. — The  trypanosomes  of  sleeping  sickness  can  be  kept  alive  for  4  or 
5  days  in  blood  mixed  with  normal  saline  solution  and  kept  at  room  tem- 
perature. In  rabbit-blood-agar  the  organisms  live  longer  but  no  true  cultures 
have  been  obtained  (Laveran  and  Mesnil). 

Experimental  inoculation. — Monkeys,  rats,  guinea-pigs, 
rabbits,  dogs,  asses,  horses,  goats,  sheep,  etc.  are  all  susceptible 
to  inoculation  with  Trypanosoma  gambiense. 

Only  certain  species  of  monkeys  are,  however,  susceptible 
(Macacus  rhesus,  M.  cynomolgus,  Cercopithecus  callitrichus,  C. 
ruber,  C.  sphinx,  etc.).  The  Cynocephali  seem  to  be  immune: 
exceptions  have  however  been  recorded  by  Thomas  and  Breinl. 
The  disease  which  follows  sub-cutaneous  or  intra-venous 
inoculation  of  the  parasite  into  susceptible  animals  is  very 
similar  to  human  trypanosomiasis.  Sleeping  sickness  has 
followed  intra-spinal  inoculation,  the  incubation  period  vary- 
ing from  10-45  days. 

Brumpt  inoculated  a  Macacus  cynomolgus  with  T.  gambiense 
with  the  result  that  the  monkey  died  in  5  weeks  with  all  the 
symptoms  of  sleeping  sickness.  Numerous  trypanosomes  were 
found  in  the  blood  :  they  were  strongly  agglomerated  by  mixing 
the  blood  with  an  equal  volume  of  potassium  citrate  solution  or 
with  serum  from  an  horse,  bovine  animal,  dog  or  man. 

Bruce  was  able  to  infect  monkeys  (Macacus  rhesus  and  Cerco- 
pithecus}   by    inoculating    them    either    sub-cutaneously,    intra- 
venously  or  intra-spinally.     Following  the  inoculation  parasites 
Stained  by  Laver-    were  found  in  the  blood,  then  symptoms  of  disease  appeared  and 
an's  method  (After    death  took  place  in  from  2-5  months.     In  a  few  of  the  monkeys 
inoculation  was  followed  by  the  appearance  of  parasites  in  the 
blood  but  the  animals  recovered  without  showing  any  symptoms. 

White  rats  can  be  easily  infected  and  most  readily  by 
intra-peritoneal  inoculation.  Trypanosomes  appear  in  the  blood  about  a 
fortnight  after  inoculation.  The  disease  lasts  about  3  months :  trypanosomes 
are  found  in  large  numbers  in  the  later  stages.  The  animals  often  recover 
and  some  of  them,  but  by  no  means  all,  are  immune. 

Dogs  and  cats  are  easily  infected  and  the  disease  proves  fatal,  as  a  rule, 
in  5-6  weeks. 


niuMagnflcation 
about    2000    dia 


820  THE   FLAGELLATA 

In  most  mammals  (guinea-pigs,  mice,  goats,  rabbits,  cattle,  horses,  etc.) 
the  trypanosome  multiplies  in  the  blood,  but  the  course  of  the  disease  is 
very  slow  and  frequently  ends  in  recovery.  The  presence  of  trypanosomes 
in  the  blood  is  often  unaccompanied  by  symptoms  (Button  and  Todd). 

^Etiology.— Bruce  and  Nabarro  showed  that  human  trypanosomiasis  and 
one  of  the  tse-tse  flies  (Glossina  palpalis}  have  a  very  similar  distribution  : 
and  they  proved  by  actual  experiment  that  this  fly  is  the  agent  by  which 
the  disease  is  spread.  In  five  experiments  flies  fed  on  patients  suffering 
from  sleeping  sickness  for  8-48  hours  infected  monkeys  (Cercopithecus). 
Similarly,  Bruce  infected  three  monkeys  by  allowing  them  to  be  bitten  daily 
over  a  long  period  by  a  large  number  of  Glossina  palpalis  collected  at  Entebbe, 
Uganda,  where  sleeping  sickness  is  prevalent. 

[The  fact  that  Glossina  palpalis  does  in  nature  transmit  the  trypanosome 
of  sleeping  sickness  is  now  in  the  region  of  facts  beyond  dispute.  But  the 
attention  of  observers  is  at  present  absorbed  in  determining  whether  other 
species  of  tse-tse  fly  can  act  as  carriers  of  the  infection  in  nature.  For  a 
while  it  was  believed  "  that  species  other  than  palpalis  were  in  this  respect 
harmless."  Certain  facts,  however,  have  since  been  disclosed  which  rendered 
a  further  investigation  into  the  natural  modes  of  transmission  imperative. 
Already  Taute  claims  to  have  demonstrated  that  Glossina  morsitans  may 
transmit  T.  gambiense  and  is  of  opinion  that  his  observations  show  that 
the  transmission  of  T.  gambiense  by  Glossina  morsitans  is  not  an  exceptional 
event.  Even  more  recently  Kinghorn  is  said  to  have  been  successful  in 
transmitting  T.  rhodesiense  (infra)  by  means  of  G.  morsitans. 

[The  tse-tse  fly  is  a  true  intermediate  host  of  the  trypanosome.  Recent 
experiments  by  Bruce  and  his  collaborators  have  shown  that  after  sucking 
infected  blood  an  incubation  period  of  28  days  follows  during  which  the 
bite  of  the  fly  is  non-infectious  and  that  at  the  end  of  that  period  the  fly  may 
become  infective  and  may  retain  its  infectivity  for  96  days  and  probably  for 
as  long  as  it  lives.  There  is  no  hereditary  transmission  of  the  parasite  from 
one  generation  of  fly  to  the  next. 

[The  source  whence  the  fly  becomes  infected  is  still  undetermined.  Bruce 
and  his  colleagues  investigating  this  point  in  the  aetiology  of  sleeping  sickness 
have  come  to  the  conclusion  that  though  no  antelope  has  up  till  the  present 
been  found  naturally  infected  with  Trypanosoma  gambiense  these  animals 
living  in  the  fly-areas  are  potential  reservoirs  of  the  virus  of  sleeping 
sickness.] 

It  is  possible  that  there  are  means  other  than  the  tse-tse  fly  by  which  human 
trypanosomiasis  is  spread.  Martin,  Leboeuf  and  Roubaud,  for  instance,  consider 
that  some  mosquitos  (Stegomyia,  Shansonia)  may  take  a  part  in  the  propagation  of 
the  disease  :  and  certain  facts  observed  in  Uganda  make  it  possible  that  the  disease 
in  man  may  be  transmitted  during  coitus. 

B.  Trypanosoma  rhodesiense.1 

Trypanosoma  rhodesiense  was  first  observed  by  J.  W.  W.  Stephens  early  in 
1910  in  a  case  of  sleeping  sickness  in  an  European  from  Northern  Rhodesia 
who  was  being  treated  in  the  Royal  Southern  Hospital,  Liverpool.  The 
trypanosome  was  described  by  Stephens  and  Fantham.2 

Stephens  and  Fantham  believe  that  sleeping  sickness  in  Rhodesia,  Nyasa- 
land,  and  adjoining  territories  is  due  to  T.  rhodesiense  and  not  to  T.  gambiense 
recently  introduced.  They  further  believe  that  T.  rhodesiense  has  existed  in 
the  territories  above  mentioned  from  time  immemorial. 

1  This  section  has  been  added. 

2  Stephens  and  Fantham,  Proc.  Roy.  Soc.  (1910),  B,  Ixxxiii  p.  28. 


THE   TRYPANOSOMES   OF  SLEEPING  SICKNESS        821 

Morphology.— T.  rhodesiense  is  characterized  by  the  fact  that  in  the  blood  of 
all  sub-inoculated  animals  the  nucleus  instead  of  being  in  the  middle  or  near 
the  middle  of  the  trypanosome,  as  is  usually  the  case,  is,  in  some  of  the  short 
or  stumpy  forms  near  the  posterior  end  more  or  less  close  to  the  blepharoplast 
or  even  on  its  posterior  side.  Stephens  and  Fantham  regard  this  morpho- 
logical feature  as  in  itself  sufficient  to  distinguish  T.  rhodesiense  from  T. 
gambiense. 

Experimental  inoculation. — T.  rhodesiense  is  distinguished  from  T.  gambiense 
by  its  great  virulence  for  the  majority  of  animal  species. 

For  rats  and  mice  T.  rhodesiense  invariably  proves  fatal  whereas  the  viru- 
lence of  T.  gambiense  for  these  species  is  subject  to  considerable  variation. 

In  guinea-pigs,  dogs  and  Macacus  monkeys  the  duration  of  T.  rhodesiense 
infections  is  shorter  than  that  of  T.  gambiense  infections. 

In  sheep  and  goats  the  difference  in  the  evolution,  symptomatology  and 
gravity  of  the  two  infections  is  quite  remarkable.  T.  rhodesiense  infections 
lead  to  an  acute  disease  with  high  fever,  oedema  and  keratitis  which  invariably 
proves  fatal  after  a  relatively  short  duration.  T.  gambiense  infections  in  these 
animals  often  give  rise  to  no  symptoms  except  fever  and  usually  end  in 
recovery. 

Cross  immunity  experiments. — Mesnil  and  Ringenbach  immunized  a  Macacus 
rhesus  against  T.  gambiense  and  then  inoculated  it  with  T.  rhodesiense.  The 
monkey  died  in  27  days.  A  control  died  in  10J  days. 

Laveran  immunized  a  goat  and  a  mouse  against  T.  gambiense  ;  when  they 
had  acquired  a  solid  immunity  they  were  inoculated  with  T.  rhodesiense. 
They  became  infected  like  the  controls. 

Laveran  and  Nattan-Larrier  immunized  a  goat  against  T.  brucei  ;  it  became 
infected  subsequently  with  T.  rhodesiense. 

Laveran  immunized  a  ram  and  a  sheep  against  different  strains  of  T.  brucei. 
On  inoculation  with  T.  rhodesiense  they  both  acquired  acute  infections  and  died. 

The  converse  series  of  experiments  are  difficult  to  effect  by  reason  of  the 
virulence  of  T.  rhodesiense ;  but  the  results,  so  far  as  they  go,  seem  to  show 
that  an  animal  immunized  against  T.  rhodesiense  is  immune  to  both  T. 
rhodesiense  and  T.  gambiense.  This  fact  according  to  Mesnil  and  Leger  does 
not  invalidate  the  specificity  of  T.  rhodesiense  but  tends  to  show  that  it  is 
closely  related  to  T.  gambiense. 

Serum  reactions.  Action  of  immune  serum. — A  goat  was  infected  with  T. 
rhodesiense.  Twenty-two  days  later  some  of  its  serum  was  mixed  with 
T.  rhodesiense  and  injected  into  a  mouse.  The  animal  survived.  A  mixture 
of  another  portion  of  the  same  serum  and  T.  gambiense  inoculated  into  another 
mouse  resulted  in  infection  (Mesnil  and  Ringenbach). 

Action  of  baboon  serum. — The  inoculation  of  1  c.c.  of  baboon  (Papio  anabis) 
serum  cured  mice  infected  with  T.  rhodesiense.  The  same  serum  given  in  the 
same  dose  had  very  little  effect  on  T.  gambiense  (Mesnil  and  Ringenbach). 

Action  of  human  serum. — In  doses  of  1  c.c.  human  serum  cured  T.  rho- 
desiense mice  in  three  cases  out  of  four.  On  T.  gambiense  human  serum  had 
no  appreciable  effect  (Stephens  and  Fantham). 

Trypanolytic  reactions. — The  serums  of  animals  (man,  monkeys  and  guinea- 
pigs)  infected  with  T.  gambiense  are  trypanolytic  for  the  homologous  trypano- 
some (T.  gambiense)  but  have  no  action  on  the  heterologous  trypanosome 
(T.  rhodesiense). 

^Etiology. — Both  in  laboratory  experiments  and  in  nature  T.  rhodesiense  is 
transmitted  by  Glossina  morsitans  (Kinghorn  and  Yorke).  In  Northern 
Rhodesia  about  16  per  cent,  of  the  wild  game  examined  are  infected  with 
T.  rhodesiense  (Kinghorn  and  Yorke). 


822  THE  FLAGELLATA 

8.  Trypanosoma  cruzi.1 

Schizotrypanum  cruzi. 

When  engaged  upon  an  anti-malarial  campaign  in  the  State  of  Minas  Geraes 
in  Brazil  in  1909  Chagas  encountered  a  large  biting  insect  known  to  the 
inhabitants  as  Barbeiro.  This  insect  which  belongs  to  the  Reduviidce  (Con- 
orrhinus  megistus)  is  about  an  inch  or  more  long  and  lives  in  cracks  in  the  walls 
or  ceilings  of  human  dwellings  from  which  it  only  emerges  in  the  darkness. 
Chagas  dissected  some  of  these  bugs  and  found  crithidial-like  flagellates  in 
the  hind  gut.  A  number  were  sent  to  Cruz  who  fed  them  upon  a  small  striped 
monkey  (Callithrix  penicillata).  Three  to  four  weeks  later  trypanosomes  of 
unusual  appearance  were  found  in  the  monkey's  blood.  Chagas'  attention 
was  now  drawn  to  a  disease  affecting  chiefly  children  of  which  the  symptoms 
were  extreme  anaemia,  enlargement  of  the  superficial  lymphatic  glands, 
oedema,  enlargement  of  the  spleen  and  functional  disturbances  especially  of 
the  nervous  system  with  frequent  occurrence  of  actual  imbecility.2  In  the 
peripheral  blood  of  one  of  these  cases  Chagas  found  a  trypanosome  which  was 
identical  with  that  seen  in  laboratory  animals  infected  with  Conorrhinus 
megistus. 

Experimental  inoculation. — Monkeys  (Callithrix),  dogs,  rabbits,  and  guinea- 
pigs  are  all  susceptible  to  infection  ;  Callithrix  and  guinea-pigs  being  more 
susceptible  than  dogs  and  rabbits.  The  disease  can  be  reproduced  either  by 
inoculation  or  by  allowing  infected  bugs  to  feed  upon  the  animal. 

Guinea-pigs  die  in  5  to  10  days.  In  the  majority  of  cases  the  trypanosome 
is  not  found  in  the  peripheral  blood  but  in  the  lungs. 

In  inoculated  monkeys  (Callithrix)  trypanosomes  appear  in  the  blood  in 
about  a  week.  The  animals  survive  six  weeks  or  so. 

Morphology.  Staining  reactions. — For  staining  blood  films  Chagas  used 
Giemsa's  solution  or  Rosenbusch's  stain. 

Rosenbusctis  stain. — Fix  the  films — whether  spread  with  blood  or  with  the  water 
of  condensation  from  blood-agar  tubes — before  they  are  quite  dry  in  Schaudinn's 
perchloride  solution  in  the  cold.  Wash  in  50  per  cent,  alcohol  then  in  water.  Treat 
for  at  least  1  hour  and  a  half  in  3 '5-5  per  cent,  iron  alum.  Stain  for  5  minutes  or 
more  in  the  following  solution  : 

1  per  cent,  solution  of  hsematoxylin  in  96  per  cent,  alcohol. 
Saturated  aqueous  solution  of  carbonate  of  lithium. 

Add  the  latter  to  the  former  until  a  wine  colour  is  obtained. 

Differentiate  with  a  very  dilute  solution  of  iron  alum  (this  operation  must  be 
watched  under  the  microscope).  Wash,  dehydrate  and  mount  in  balsam. 

In  the  peripheral  blood. — In  the  peripheral  blood  the  parasite  may  be 
within  the  red  cells,  partly  within  the  red  cells,  merely  attached  to  the  red 
cells  by  the  blepharoplast  or  may  be  free  in  the  plasma.  The  parasites  exhibit 
a  sexual  dimorphism.  The  so-called  male  parasites  are  relatively  slender,  have 
an  elongated  nucleus,  a  strikingly  large  blepharoplast  and  often  exhibit  a 
second  mass  of  chromatin  in  front  of  the  nucleus.  The  so-called  female 
parasites  are  somewhat  short  and  squat,  the  blepharoplast  is  situated  termin- 
ally or  very  near  the  end  and  the  nucleus  consists  of  a  loose  mass  of  chro- 
matin. These  appearances  are  very  similar  alike  in  man,  monkeys  and  guinea- 
pigs. 

In  the  lungs. — Multiplication  does  not  take  place  in  the  circulating  blood. 
In  the  lungs  certain  multiplication  forms  are  seen  which  Chagas  regards  as 

1  This  section  has  been  added. 

2  The  symptoms,  and  especially  the  dropsy  and  nervous   symptoms  which  precede 
death,  are  so  like  the  symptoms  of  ankylostomiasis  that  the  disease  is  known  locally 
as  Opilacao  and  Canguary—the  names  given  to  ankylostomiasis. 


THE  TRYPANOSOME   OF  CHAGAS'  DISEASE  823 

gametogony — in  contradistinction  to  what  he  believes  to  be  simple  schizogony 
taking  place  in  other  parts  of  the  body  (vide  infra). 

In  the  lungs  the  trypanosome  loses  its  undulating  membrane,  the  two  ends 
curve  towards  each  other  like  a  crescent  and  unite  ;  the  female  parasites 
then  shed  the  blepharoplast  and  in  both  the  male  and  female  parasites  the 
chromatin  divides  into  eight  secondary  nuclei  giving  rise  to  eight  merozoites, 
those  derived  from  female  parasites  having  a  single  nucleus,  the  others  having 
both  a  nucleus  and  a  blepharoplast  connected  by  a  fine  thread  of  chromatin. 
These  merozoites  (the  precursors  of  the  gametes  found  in  the  circulating 
blood)  then  enter  a  red  cell  and  develop  into  typical  trypanosomes.  These 
forms  have  been  found  in  man,  monkeys  (Callithrix),  cats  and  dogs  but  are 
very  uncommon  in  guinea-pigs. 

To  demonstrate  the  changes  above  described  Chagas  recommends  that  a  guinea- 
pig  should  be  infected  with  a  Conorrhinus  and  that  1-2  c.c.  of  the  blood  of  this  first 
animal  should  be  inoculated  intra-peritoneally  into  a  second  guinea-pig  which 
should  be  killed  5  or  6  days  later. 

In  other  structures. — Within  the  cells  of  certain  other  tissues,  and  notably 
in  cardiac  and  striated  muscular  tissue  and  in  neuroglia  cells,  the  trypanosome 
multiplies,  according  to  Chagas,  by  simple  schizogony  and  gives  origin  to  a 
great  number  of  daughter  parasites  each  having  a  nucleus  and  centrosome. 
In  the  cells  of  the  central  nervous  system  the  young  trypanosomes  may 
proceed  to  the  flagellated  stage.  The  infected  host  cell  is  reduced  to  a  mere 
envelope  and  the  contents  with  the  exception  of  the  nucleus  are  destroyed. 

In  the  insect  carrier. — In  the  mid-gut  of  Conorrhinus  megistus  the  blepharo- 
plast approaches— and  perhaps  blends  with — the  nucleus,  the  undulating  mem- 
brane disappears,  the  parasite  becomes  rounded  and  then  multiplies  rapidly 
by  division.  The  daughter  parasites  become  flagellated,  the  flagellum  taking 
origin  from  the  blepharoplast.  In  the  posterior  cylindrical  portion  of  the 
mid-gut  numerous  flagellated  crithidial  forms  are  found. 

On  two  occasions  Chagas  found  trypanosomes  in  the  body  cavity  and  in 
the  salivary  glands  of  the  bug.  The  latter  no  doubt  represent  the  forms 
which  are  inoculated  when  the  insect  bites  a  susceptible  animal. 

Cultivation.— The  parasite  grows  easily  on  Novy  and  MacNeaPs  blood- 
agar.  The  cultivation  forms  are  similar  to  the  forms  found  in  the  bug — 
round  forms,  rapidly  dividing  pear-shaped  forms  and  crithidial  forms.  The 
parasite  can  almost  always  be  sub-cultivated  twice. 

Etiology. — Chagas  concludes  that  the  bug  Conorrhinus  megistus  does  not 
play  a  purely  mechanical  part  in  the  transmission  of  American  trypanosomiasis. 
The  bug  is  not  infective  for  at  least  a  week  after  the  infecting  meal. 

In  Chagas'  opinion  there  are  two  different  forms  of  development  in  the  bug. 
One — the  last  stage  of  which  is  represented  by  the  crithidial  forms  in  the  mid- 
gut  and  which  is  always  seen  after  the  insect  is  fed  on  infected  blood — is 
without  importance.  The  other  which  is  very  imperfectly  known  probably 
represents  the  true  cycle. 

Detection  of  the  parasite. — Blood  films  may  be  prepared  and  stained  by 
Giemsa's  and  Rosenbusch's  method  but  it  is  better  to  inoculate  the  blood  into 
the  peritoneal  cavity  of  a  guinea-pig. 

9.  Trypanosomes  in  birds. 

Danilewsky  [in  1888]  was  the  first  to  give  a  description  of  trypanosomes 
in  birds  (Trypanosoma  avium).  Kecent  work  has  shown  that  there  are 
several  species  of  avian  trypanosomes  (Laveran,  Button  and  Todd,  Hanna). 

A  large  number  of  birds  are  known  to  be  infected  ;  for  instance  owls,  roller- 


824 


THE   FLAGELLATA 


birds,  pigeons,  Indian  crows,  the  chaffinches,  the  gold  finches,  Java  sparrows 
(Padda  oryzivora).  The  parasites  are  present  in  the  blood  and  bone  marrow. 

[Petrie  found  trypanosomes  in  several  species  of  birds  at  Elstree  in  Hert- 
fordshire— house  martins,  song  thrushes,  blackbirds,  swallows,  yellow- 
hammers.  The  trypanosomes  were  not  found  in  the  blood  but  only  in  the 
bone  marrow.] 

The  following  description  by  Danilewsky  revised  by  Laveran  is  applicable  to  the 
Trypanosoma  avium  [of  the  owl,  Syrnium  aluco].  The  parasite  is  fusiform  in  shape 
and  has  an  undulating  membrane  and  an  anterior  flagenum.  The  cytoplasm  stains 
deeply  by  Laveran's  method,  so  deeply  that  the  tropho -nucleus  and  kineto-nucleus 
are  only  just  visible.  The  trypanosome  including  the  nagellum  is  about  S3-45// 
long.  Multiplication  takes  place  by  longitudinal  fission. 


FIG.  397—  Bird  trypanosomes.     (After  Danilewsky.) 


The  trypanosome  can  live  5-8  days  in  blood  kept  aseptically  in  a  pipette  at  a 
temperature  of  22°  C.  Under  these  conditions  Danilewsky  has  observed  spherical 
bodies  which  divide  and  give  rise  to  spherical  amoeboid  bodies  each  having  a  nucleus  : 
these  bodies  become  pyriform  and  a  very  motile  flagellum  appears  at  their  anterior 
extremities  (Trypanomonas,  Danilewsky).  After  a  certain  lapse  of  time  these  new 
forms  assume  the  characteristic  appearances  of  the  trypanosome. 

In  addition  to  T.  avium  other  trypanosomes  are  found  in  birds,  e.g.  trypano- 
somes of  the  type  T.  rotatorium  of  frogs,  and  long  slender  trypanosomes  with 
no  free  flagellum. 

10.  Trypanosomes  in  cold  blooded  vertebrata. 

Trypanosomes  have  been  found  in  Batrachians,  reptiles  and  fish. 

Trypanosomes  in  frogs. — Many  species  of  trypanosomes  are  found  in  frogs.  The 
most  widely  distributed  is  Trypanosoma  rotatorium  (Undulina  ranarum  [Ray  Lan- 
kester],  Trypanosoma  sanguinis  [Gruby])  which  ha?  been  studied  by  Gliige,  Danilew- 
sky, Mayer,  Gruby,  Chalachnikow,  Ray  Lankester  and  others.  It  is  found,  especially 
in  summer,  in  Rana  esculenta,  R.  viridis,  Hyla  viridis,  Bufo  vulgaris,  etc. 

T.  rotatorium  varies  much  in  size  and  shape.  There  is  a  flat  form  enrolled  on 
itself ;  a  simple  flat  form,  membranous,  very  active ;  pectinated  forms  either 
fan- shaped  or  in  the  form  of  a  cornucopia,  etc.  In  length  it  varies  from  40-60/x 
and  even  75/x,  and  in  width  from  5-40/z.  It  is  the  largest  of  all  the  known  trypano- 
somes (Laveran  and  Mesnil). 

The  cytoplasm  contains  a  nucleus  and  a  centrosome  always  situated  close  together, 
the  nucleus  being  anterior  to  the  centrosome.  The  undulating  membrane  is  very 
much  folded  and  its  free  border  is  continued  anteriorly  as  the  flagellum. 

Trypanosomes  in  Fish. — Trypanosomes  were  discovered  in  fish  by  Valentin  in 
the  blood  of  [a  trout]  Salmofario,  and  they  have  since  been  found  by  Remak,  Mitro- 
phanow,  Danilewsky,  Chalachnikow,  Lingard  and  others  in  [a  loach]  Cobitisfossilis, 
[the  Prussian  carp]  Carassius  vulgaris,  [carp]  Cyprinus  car  pis,  Perca  fluvialitis,  and 
other  fish. 

Laveran  and  Mesnil  have  found  Trypanosomes  in  sea  fish  :  [ray]  Raja  punctata, 
R.  mosaica;  [small  dog-fish]  Scyllium  canicula,  and  [large  dogfish]  Sc.  stellare  ; 
[sole]  Solea  vulgaris. 


TKICHOMONAS  VAGINALIS  825 

Chalachnikow  distinguishes  two  varieties  of  trypanosomes  in  fish  ;  one  similar 
to  T.  avium,  the  other,  flat  and  not  folded,  like  T.  rotatorium.  Numerous  species 
are  now  described :  T.  remaki,  T.  danilewskyi,  T.  dbramis,  T.  rajce,  T.  solece,  etc. 


FIG.  398. — Fish  trypanosomes.    (After  Chalachnikow.) 

For  the  detection  of  the  parasites  in  the  blood,  Laveran  and  Mesnil  advise  col- 
lecting a  few  drops  of  blood  by  incising  two  or  three  rays  at  the  base  of  the  dorsal 
fin.  The  blood  should  be  examined  between  a  slide  and  a  cover-glass.  Mixing  the 
blood  with  a  little  citrated  normal  saline  solution  prevents  coagulation  and  preserves 
the  motility  of  the  parasites. 

For  preparing  stained  preparations  the  living  fish  must  be  opened  and  some  of 
the  heart  blood  collected  in  a  pipette  :  the  blood  is  then  to  be  spread  on  slides  in  a 
thin  layer,  rapidly  dried  over  the  flame  of  a  spirit  lamp  and  then  fixed  in  absolute 
alcohol  or  in  alcohol-ether. 


SECTION  II.— TRICHOMONAS  VAGINALIS. 

Syn. — Cercomonas  intestinalis.     Trichomonas  intestinalis. 

It  is  now  generally  conceded  that  the  parasites  described  under  the  names 
of  Cercomonas  hominis  (Davaine),  Cercomonas  intestinalis  (Lambl),  Tricho- 
monas intestinalis  (Leuckart),  etc.,  should  be  regarded  as  a  single  species  : 
Trichomonas  vaginalis  (Donne,  Blanchard). 

Trichomonas  vaginalis  was  discovered  by  Davaine  in  the  dejecta  of  persons  suffering 
from  cholera.  It  is  frequently  to  be  found  in  the  stools  of  patients  suffering  from 
diarrhoea  due  to  very  different  diseases.  It  has  been  found  in  the  viscous  fluid  sur- 
rounding an  hydatid  cyst  of  the  liver  (Lambl),  in  cases  of  gangrene  of  the  lung, 
hydropneumothorax,  etc.  It  is  frequently  present  in  the  vagina  in  cases  of  vaginitis 
in  married  women  and  virgins,  but  not  in  normal  alkaline  vaginal  mucus.  It  has 
been  found  in  the  bladder. 

In  some  cases  of  dysentery  Trichomonas  is  found  in  very  large  numbers  (Castellani, 
Terry,  Chassin,  Billet)  and  may  be  found  in  association  with  Amoeba  histolytica. 
The  part  it  plays  in  disease  processes  is  not  clear  :  there  is  no  proof  that  alone  it  can 
give  rise  to  dysenteriform  ulceration.  It  is  conceivable  that  it  may  find  a  suitable 
soil  for  development  in,  and  may  perpetuate,  some  pre-existing  lesion. 

According  to  Perron9ito,  the  parasite  gains  entrance  to  the  alimentary  canal  in 
drinking  water,  in  which  it  occurs  in  an  encysted  form. 

Morphology. — Trichomonas  vaginalis  measures  12-25//,  long  by  7-1 2/* 
broad.  Its  shape  is  very  variable  ;  it  is  generally  pyriform,  though  some- 
times pointed,  sometimes  curled  up  into  a  rounded  ball,  and  sometimes 
constricted  in  the  centre  like  an  hour-glass.  The  larger  anterior  end  is 
furnished  with  three  sessile  flagella  directed  forwards  all  attached  at  the 
same  point  and  frequently  matted  together  :  there  is  a  fourth  flagellum  turned 
backwards  forming  the  free  margin  of  a  slightly  raised,  folded  and  scalloped 
undulatory  membrane  which  extends  to  the  posterior  end  of  the  organism. 
The  posterior  end  is  most  frequently  provided  with  a  caudal  appendage  of 


826 


THE  FLAGELLATA 


variable  length  :  the  mouth  opens  near  the  insertion  of  the  flagella  and  leads 
to  a  tubular  cavity  in  the  protoplasm. 

The  rounded  and  elongated  nucleus  and  the  blepharoplast  are  situated  in 
the  granular  protoplasm  near  the  mouth.  A  slender  hyaline  rod  is  seen  near 
the  middle  of  the  body. 

The  shape  of  the  parasite  is  subject  to  considerable  variation  and  on  occa- 
sions it  throws  out  pseudopodia.  In  the  intestine  (mucus  from  a  case  of 
dysentery)  Ballet  has  seen  a  number  of  Trichomonas  agglutinated  together 


FIG.    400. — A,    Cercomonas    intestinalis 
(after  Davaine) ;  B,  Trichomonas  vaginalis. 
FIG.  399. — Trichomonas  vaginalis.  In  this  figure  A  and  B  are  really  iden- 

(After  Grassi.)  tical.   In  A,  the  parasites  show  only  a  single 

flagellum  the  others  have  been  accidentally 
destroyed.  The  flagella  pictured  on  one  of 
the  parasites  in  B  are  merely  artefacts  due 
to  the  tearing  of  the  undulating  membrane. 

in   a   regular-shaped  rosette,  the  individuals  forming  which  soon  become 
changed  into  large,  vacuolated,  slightly  motile,  amosboid  masses  each  having 
a  nucleus  placed  excentrically. 
Reproduction  is  by  longitudinal  division. 

Methods  of  examination. — Examination  of  the  parasite  is  difficult  on 
account  of  its  extreme  motility  and  the  details  of  its  structure  can  only  be 
studied  after  fixing  by  one  of  the  methods  described  when  dealing  with  the 
amoabse. 

Search  must  be  made  for  T.  intestinalis  in  stools  while  they  are  still  warm, 
as  the  parasite  quickly  dies  when  they  cool.  In  carrying  out  this  investiga- 
tion all  the  precautions  mentioned  when  dealing 
with  the  amoebae  must  be  observed,  and  if  need 
be  the  stools  should  be  diluted  with  luke-warm 
normal  saline  solution  or  Grassi's  solution. 

Other  species  of  Trichomonas. 
Cercomonas  termo. — Cercomonas  termo  is  an  excellent 
species  for  the  study  of  these  Protozoa.     It  occurs 
in  large  numbers  in  vegetable  infusions. 

Cercomonas  termo  consists  of  an  oval  body  having 
at  one  end  a  long,  very  motile  flagellum. 

Food  stuffs  are  introduced  into  the  protoplasm  at 
the  cytostome  or  mouth  situated  at  the  base  of  the 
flagellum ;  at  this  point  the  ectoplasm  is  interrupted 
and  the  protoplasm  is  vacuolated;  food  stuffs  are  collected  in  the  vacuole  which 
extends  to  the  centre  of  the  body,  and  here  the  ingested  particles  are  digested. 
Foreign  bodies  other  than  food  particles  which  may  hive  been  ingested  are  rejected 

ame  point  at  which  they  were  absorbed  (Biitschli) 
Reproduction  is  by  binary  longitudinal  fission.     The  nucleus  divides  first  and  is 


FIG.  401. — Cercomonas  termo. 


LAMBLIA  INTESTINALIS  827 

followed  by  division  of  the  protoplasm  in  the  neighbourhood  of  the  flagellum :  the 
two  daughter  cells  gradually  separate  and  a  flagellum  develops  on  the  part  which 
has  been  deprived  of  it. 

Trichomonas  c  a  vise. — This  parasite  is  responsible  for  certain  epizootic  diseases 
among  guinea-pigs  (Galli- Valeric).  The  protozoon  is  found  in  large  numbers  on 
the  epithelial  surface  of  the  intestine. 

Trichomonas  batracorum. — T.  batracorum  lives  in  the  intestines  of  frogs.  It  is 
elongated  and  spindle-shaped,  the  anterior  end  being  larger  than  the  posterior  and 
provided  with  two  or  three  flagella.  The  posterior  end  has  a  long  flagellum  attached 
laterally.  The  undulating  membrane,  the  free  edge  of  which  is  serrated  like  the 
teeth  of  a  saw,  extends  from  the  anterior  extremity  to  the  base  of  the  posterior 
flagellum. 

Monas  pyophila. — This  parasite  has  been  found  in  the  pus  of  a  liver  abscess  in 
Japan  by  Grimm.  In  appearance  it  is  like  that  of  a  large  spermatozoon  (30-60/x 
long)  prolonged  at  one  end  in  the  form  of  a  long  appendix  which  terminates  in  a 
flagellum. 

Bodo  urinarius. — This  flagellate  grows  easily  in  alkaline  urines ;  it  appears  to  be 
an  harmless  organism  and  probably  occurs  in  the  encysted  form  in  atmospheric  dust, 
and  gains  access  to  urine  after  the  latter  has  been  passed.  It  has  been  described  by 
Hassal,  Salisbury,  Kiinstler,  Barrois  and  others  and  is  variously  known  as  B.  urina- 
rius, Plagiomonas  irregularis,  PI.  urinaria,  Cystomonas  urinaria. 

It  has  an  oval-shaped  protoplasmic  body  (12-15/xx  about  8/>t),  the  anterior  end 
being  the  larger  and  carrying  two  flagella :  the  posterior  end  is  prolonged  into  an 
elongated  retractile  point. 


SECTION  III.— LAMBLIA  INTESTINALIS. 

Synonym. — Megastoma  entericum. 

This  parasite  was  discovered  by  Lambl  in  the  mucus  in  the  stools  of  young 
children.  It  has  since  been  frequently  found  in  the  stools  or  intestinal 
contents  of  persons  in  good  health  and  in  others  affected  with  various 
diseases,  and  especially  in  young  children  and  persons  suffering  from  tuber- 
culous phthisis  :  it  inhabits  preferably  the  duodenum  and  jejunum.  Noc 
has  recorded  its  occurrence  in  the  stools  of  persons 
suffering  from  Cochin  China  diarrhoea.  LambliaB  do 
not  appear  to  be  pathogenic.  Sometimes  they  are  so 
numerous  as  to  cover  a  large  part  of  the  mucous 
membrane  of  the  small  intestine ;  in  a  patient  affected 
with  chronic  gastric  catarrh  Moritz  and  Holzl  estimated 
the  number  of  parasites  evacuated  in  24  hours  to  be 
18  thousand  millions.  Lamblise  are  also  found  in  the 
dog,  cat,  sheep,  rabbit,  rat,  mouse,  etc. 

Morphology. — Lamblia  intestinalis  is  pyriform  in 
shape  and  measures  10-20//,  long  by  5-10/x  broad. 
On  one  side  of  the  larger  extremity  it  presents  a  cup- 
shaped  depression.  There  are  four  pairs  of  flagella  ;  FIG.  402.— Lamblia  intes- 
the  first  pair  take  origin  from  the  anterior  extremity,  fSiakoln*  Grassi  and 
the  second  and  third  pairs  from  the  posterior  end 

of  the  depression  while  the  fourth  pair  are  attached  to  the  pointed  posterior 
end  of  the  parasite  :  these  flagella  measure  7-1 4/x  long,  are  directed  back- 
wards and  are  capable  of  varied  movement. 

The  protoplasm  is  generally  granular  :  it  possesses  a  nucleus  situated 
transversely  and  having  the  shape  of  a  dumb-bell  or  an  horse-shoe. 

In  the  intestine  the  parasite  occurs  on  the  surface  of  the  villi  and  attaches 
itself  by  means  of  the  sucker  to  the  epithelial  cells,  its  posterior  extremity 
being  then  vertical  to  the  surface  or  directed  forwards.  In  the  intestine 


828  THE   FLAGELLATA 

Lamblia  multiplies  by  fission.  Dissemination  is  effected  by  means  of  oval 
cysts  which  are  discharged  in  the  stools,  infection  taking  place  through 
drinking  water  or  foods  infected  with  the  cysts.  Calandruccio  infected  him- 
self by  swallowing  a  number  of  cysts  recovered  from  the  stools  of  an  infected 
person  :  mice  and  rats  ( Mus  decumanus)  have  been  infected  by  the  same 
means  (Perrongito,  Grassi). 

Methods  of  detection. — The  technique  to  be  adopted  is  that  described  for 
the  amoebae. 


CHAPTER  LXIII. 
THE  INFUSORIA. 

Introduction  and  methods  of  examination. 
Parasitic  species,  p.  830. 

THE  Infusoria  are  Protozoa  the  bodies  of  which  are  wholly  or  in  part  covered 
with  motor  appendages  or  cilia.  They  have  a  distinct  ectoplasm  and  endo- 
plasm, contractile  fibres,  contractile  vacuoles,  and  a  nucleus  divided  into 
two  parts  (macronucleus  and  micronucleus).  In  addition  they  have  a  mouth, 
a  pharynx  opening  into  the  endoplasm  and  an  anus  which  is  only  visible  at 
the  moment  of  expulsion  of  the  excreta. 

They  multiply  generally  by  transverse  division.  After  several  generations 
have  been  produced  by  fission  the  individuals  show  a  tendency  to  conjugate, 
and  after  certain  changes  have  taken  place  in  the  nucleus  of  the  two  con- 
jugated individuals  the  latter  separate  and  each  may  then  become  the  parent 
of  a  further  generation  of  schizogonic  elements. 

In  the  stools  and  outside  the  body  cysts  are  produced  :  the  animal  loses 
its  cilia,  becomes  rounded  and  appears  to  consist  of  a  dark  central  mass 
surrounded  by  a  clearer  peripheral  zone.  The  cyst  is  the  latent  form  of  the 
organism  which  again  becomes  ciliated  when  conditions  are  favourable. 

Five  species  of  Infusoria  are  known  which  occur  as  parasites  in  man. 

Methods  of  examination. — The  technique  to  be  adopted  is  similar  to  that 
described  in  the  case  of  the  Amoebae.  For  detailed  study  of  their  structure 
it  will  be  found  useful  to  stain  the  Infusoria  in  the  living  state,  either  with  a 
solution  of  quinolein  blue  (which  stains  the  granules  of  the  endoplasm  but 
neither  the  nucleus  nor  the  cilia)  or  with  Bismarck  brown  (which  stains  the 
vacuoles  first  then  the  protoplasm  but  leaves  the  nucleus  unstained).  Violet 
dahlia  or  malachite  green  may  be  used  for  staining  the  nucleus.  The 
dyes  should  be  dissolved  in  the  fluid  in  which  the  Infusoria  are  living 
(normal  saline  solution)  and  the  solutions  should  be  weak  (about  1  in 
10,000). 

For  fixing  the  Infusoria,  osmic  acid  gives  the  best  results.  The  vapour 
of  the  acid  should  be  allowed  to  act  on  the  slide  on  which  the  drop  of  water 
containing  the  parasites  is  placed  :  or  a  drop  of  a  1  per  cent,  solution  of 
osmic  acid  may  be  placed  on  a  cover-glass  and  inverted  on  to  the  water  on 
the  slide. 

Tissues  intended  for  histological  examination  should  be  fixed  in  Flemming's 
solution  or  in  30  per  cent,  formalin  :  the  methods  of  staining  are  the  same 
as  for  the  Amoabae. 


830 


THE   INFUSORIA 


Balantidium  coli. 

(Paramoecium  coli.) 

This  protozoon  is  found  in  the  intestines  and  stools  of  man  and  pigs  :  it 
was  first  described  by  Malmsten  but  it  appears  to  have  been  previously  seen 
by  Leeuwenhoek  in  the  excreta. 

It  would  seem  to  have  been  proved  that  Balantidium  coli  may  produce 
disease  :  it  has  been  found  in  numerous  cases  of  catarrhal  colitis  and  ulcera- 
tive  dysentery  in  man  in  America,  the  Philippines,  Finland,  Bothnia,  Russia, 
Germany,  etc.  (Russell,  Losch,  Strong  and  Musgrave,  and  others). 


FIG.  403.— Balantidium  coli. 


FIG.  404. — Balantidium  coli  in  stools.    (After  Guiart.) 


Askanazy,  and  Klimenko  have  shown  that  not  only  is  Balantidium  coli 
found  in  extraordinarily  large  numbers  in  ulcerative  lesions  of  the  intestine 
but  that  it  finds  its  way  into  the  deep  layers  of  the  colon,  into  healthy 
tissues  and  even  into  small  blood  and  lymph  vessels  :  this  power  of  penetra- 
tion explains  the  finding  of  the  parasite  by  Manson  and  Stockvis  in  liver 
abscesses.  [Brooks  attributed  a  fatal  epizootic  of  dysentery  among  the  orang- 
outangs in  the  zoological  gardens  at  New  York  to  this  parasite.] 

It  is  necessary,  however,  to  point  out  that  up  till  now  it  has  not  been 
possible  to  produce  experimentally  in  man  or  in  monkeys  an  ulcerative 
colitis  with  Balantidium  coli.  Grassi  and  Calandruccio  failed  to  infect 
themselves  by  swallowing  cysts  of  Balantidium  coli  obtained  from  a  pig. 

Morphology  .—Balantidium  coli  is  an  oval-shaped  organism  measuring 
from  70-100/x  long  which  can  be  seen  with  the  unaided  eye  :  its  surface  is 
covered  with  short,  delicate  vibratile  cilia  arranged  in  regular  longitudinal 
lines. 

At  the  narrower  end  of  the  body  there  is  a  mouth  or  cytostome  destined  to 
receive  the  food,  and  at  the  larger  end  a  second  orifice  or  cytopyge  for  the 
removal  of  the  waste  products  of  digestion.  Around  the  mouth  the  cilia 
are  grouped  in  a  -ring  or  collar  and  move  in  such  a  way  as  to  direct  food 
material  towards  the  mouth.  The  arrangement  of  the  "nucleus  is  charac- 
teristic :  the  macronucleus  has  the  shape  of  an  haricot  bean  in  the  concavity 
of  which  the  rounded  micronucleus  is  situated.  The  protoplasm  contains 
two  or  three  contractile  vacuoles  in  which  ingested  materials  of  external 
origin  such  as  blood  cells,  starch  granules,  fat  droplets,  etc.  are  frequently 
found. 

The  cysts  are  spherical  (vide  ante)  and  measure  80-100/x  across.  They 
are  found  in  cold  and  dried  excreta. 


THE  INFUSORIA 


831 


FIG.  405. — Balantidium 
minutum.  (After  Schaudinn.) 


Balantidium  minutum. 

This  parasite  was  first  discovered  by  Jacobi  and  Schaudinn  at  Strasbourg 
in  the  intestines  of  a  ship's  cook.  It  has  since  been  found  in  Berlin 
twice  (once  in  association  with  Nyctotherus  faba);  and  in  Porto  Rico  in 
several  cases  of  dysentery  together  with  Balantidium 
coli. 

It  is  pyriform  in  shape  and  only  measures  20-30/x  x 
15-20/u.  The  macronucleus  is  spherical  and  situated 
centrally,  the  micronucleus  is  attached  to  it.  There 
is  only  one  contractile  vacuole. 

Golpoda  cucullus. 

This  parasite  is  very  commonly  found  in  marshes 
and  can  live  in  the  human  intestine  :  it  was  found  by 
Schultz  in  the  intestine  of  a  person  suffering  from 
dysentery.  Morphologically  it  is  very  like  Balanti- 
dium minutum  with  which  it  is  possibly  identical. 

[Stokvis  and  Swellengrebel  have  shown  that  living 
Colpoda  cucullus  will  purify  water  of  bacteria  present 
in  it.] 

Nyctotherus  faba. 

This  parasite  was  twice  found  by  Jacobi  and  Schaudinn  once  in  Berlin  and 
once  in  Strasbourg.  It  has  the  shape  of  an  haricot  bean  :  the  mouth  occu- 
pies the  anterior  one-half  of  the  concave  side.  The  macronucleus  is  spherical 
and  has  the  micronucleus  attached  to  it.  It  has  only 
a  single  contractile  vacuole  (fig.  406). 

Chilodon  dentatus. 

This  organism  was  found  by  Guiart  in  Paris  in  the 
stools  of  a  woman  suffering  from  dysenteric  colitis. 
Its  common  habitat  is  water.  The  parasite  is  an  oval- 
shaped  Infusorium  (35-55/u,  x  25-35/x),  the  ventral  surface 
being  flattened  and  the  dorsal  markedly  convex.  At 
its  anterior  part  it  shows  a  flexible  and  membranous 
prolongation  of  the  ectoplasm  having  several  rows  of 
long  flagella  on  its  lower  surface.  The  ventral  surface 
, . [G-  ±W-— Nyctotherus  of  ^he  parasite  appears  to  be  covered  with  very  short 

faba.     (After  Schaudinn.)  r  rr  w  J 

flagella  while  the  dorsal  surface  is  bare.  I  he  moutn 
is  centrally  placed  towards  the  anterior  part  of  the  endoplasm :  its  margins 
are  covered  by  chitinous  rods.  The  macronucleus  is  rounded  and  situated 
towards  the  posterior  part  of  the  body  :  the  micronucleus  is  attached  to 
it.  There  are  two  contractile  vacuoles  one  in  front  of,  the  other  behind, 
the  nucleus. 


PART   VI. 
THE  FILTEABLE   VIRUSES. 


3G 


CHAPTER  LXIV. 
THE  FILTKABLE  VIRUSES. 

Introduction. 

Section  I. — The  virus  of  Pleuro-pneumonia  in  cattle,  p.  836. 
Section  II. — The  virus  of  Foot  and  Mouth  disease,  p.  838. 
Section  III. — The  virus  of  Horse-sickness,  p.  838. 
Section  IV. — The  virus  of  Rinderpest,  p.  839. 
Section  V.— The  virus  of  Bird-plague,  p.  839. 
Section  VI. — The  virus  of  Sheep-pox,  p.  839. 
"  The  infectious  epithelioses,"  p.  840. 
Section  VII. — The  virus  of  Cow-pox  p.  840. 
Section  VIII.— The  virus  of  Yellow  fever,  p.  841. 
Section  IX.— The  virus  of  Rabies,  p.  841. 
Section  X. — Filtrable  viruses  in  the  Pasteurelloses,  p.  842. 
Section  XI. — The  virus  of  Swine  fever,  p.  843. 
Section  XII. — The  virus  of  Acute  anterior  poliomyelitis,  p.  844. 
Section  XIII. — The  virus  of  Typhus  fever,  p.  847. 

PASTEUR  put  forward  the  opinion  that  some  micro-organisms  were  so  small 
as  to  escape  the  ordinary  method  of  microscopical  investigation  ;  recent 
research  has  proved  the  truth  of  this  hypothesis,  and  has  shown  moreover 
that  micro-organisms  too  small  to  be  seen  with  the  microscope  are  respon- 
sible for  a  number  of  diseases.  The  use  of  dark-ground  illumination  has 
not  hitherto  given  any  interesting  results  in  this  connexion. 

It  is  characteristic  of  the  filtrable  viruses  that  they  can  pass  through 
certain  porous  porcelain  or  similar  niters  and  for  this  reason  they  are  some- 
times known  as  "  filter  passers." 

To  demonstrate  this  property  too  fine  a  filter  must  not  be  used  :  a  Berke- 
feld  V  or  Chamberland  F  are  the  most  suitable  for  the  purpose  though  occa- 
sionally a  more  porous  bougie  is  employed  (Chamberland  Fj  to  F10  made  to 
the  instructions  of  Borel,  or  Berkefeld  V  worn  down  on  a  grindstone  or  with 
a  glass  cutter). 

The  virus  is  prepared  as  follows  :  Dilute  the  infected  material  (defibrinated 
blood,  serous  exudate,  emulsion  of  internal  organs,  etc.)  with  water  in  which 
a  culture  of  an  easily-recognized  micro-organism  preferably  a  motile  organism 
— has  been  emulsified  :  this  organism  acts  as  a  control.  Filter  the  emulsion 
under  a  low  pressure.  On  cultivation  the  filtrate  should  yield  no  visible 
growth  but  on  inoculation  into  a  susceptible  animal  should  give  rise  to  the 
original  disease. 

In  experiments  with  ultra-microscopic  viruses  certain  conditions  must  be  strictly 
observed.  The  filter  should  be  new  and  must  be  sterilized  before  use :  the  process 
of  filtration  should  occupy  not  more  than  about  2  hours  :  the  pressure  applied — 


836  THE   FILTRABLE   VIRUSES 

whether  by  compression  or  aspiration — should  be  as  small  as  possible  (in  the  former 
case,  say,  the  pressure  produced  by  an  india-rubber  syringe,  and  in  the  latter  that 
equivalent  to  50-500  mm.  of  mercury) :  the  experiment  should  be  carried  out 
at  the  ordinary  temperature  of  the  atmosphere  and  if  possible  not  above  20°  C.  : 
the  emulsion  should  be  diluted  so  as  to  avoid  blocking  the  pores  of  the  filter  with 
the  albuminous  matter  present :  and  finally,  since  even  those  ultra- microscopic 
viruses  which  are  most  easily  filtered  are  partially  retained  in  the  filters,  several 
animals  should  be  inoculated  each  with  a  large  volume  of  the  filtrate. 


SECTION  I.— PLEURO-PNEUMONIA   OF   CATTLE. 

[Pleuro-pneumonia  contagiosa.] 

All  attempts  to  demonstrate  the  micro-organism  of  contagious  pleuro- 
pneumonia  in  cattle  failed  until  Nocard  and  Roux  in  1898  devised  a  new 
method  of  investigation  (vide  post)  which  resulted  in  the  discovery  of  the 
organism.  "  The  discovery  of  the  cause  of  pleuro-pneumonia,"  wrote  these 
observers,  "  is  interesting  not  only  because  peculiar  difficulties  have  been 
overcome  but  because  it  affords  hope  that  a  similar  success  may  attend  the 
study  of  those  other  diseases  of  which  the  micro-organism  is  up  till  the 
present  unknown." 

Pleuro-pneumonia  of  cattle  may  run  either  an  acute  or  a  chronic  course.  In  the 
acute  form  of  the  disease  the  respiratory  symptoms  are  the  most  marked  :  the 
respiratory  movements  are  increased  in  frequency  and  are  shallow,  friction 
sounds  can  be  heard  as  well  as  rhonchi  denoting  bronchial  disease,  and  there  is  a 
frequent  cough  and  running  from  the  nose  ;  the  animal  ceases  to  chew  its  cud  and 
loses  its  appetite  :  the  disease  may  resolve,  become  chronic,  or  may  terminate 
fatally.  In  the  chronic  form  of  the  disease — which  may  either  begin  as  such  or 
follow  an  attack  of  the  acute  form — a  considerable  area  of  the  lungs  is  congested 
and  hepatized  :  as  a  rule,  the  disease  is  incurable. 

The  essential  lesion  in  pleuro-pneumonia  is  the  distension  of  the  meshes  of  the 
inter- lobular  connective  tissue  with  a  large  amount  of  clear  amber- coloured  fluid. 

1.  Experimental  inoculation.    Vaccination. 

Willens  has  shown  that  the  exudate  in  pleuro-pneumonia  will  reproduce 
the  disease  on  inoculation  into  bovine  animals.  Goats,  sheep,  pigs,  dogs, 
guinea-pigs,  rabbits  and  birds  are  immune. 

The  inoculation  of  a  drop  of  the  fresh  serous  exudate  from  a  case  of  pleuro- 
pneumonia  into  the  sub-cutaneous  tissues  of  a  cow  is  followed,  after  an 
incubation  period  of  from  8-25  days,  by  a  disease  the  severity  of  which  will 
depend  upon  the  site  of  inoculation. 

(i)  When  the  material  is  inoculated  beneath  the  skin  of  the  trunk  or  neck, 
the  temperature  of  the  inoculated  animal  becomes  very  much  raised  and  is 
accompanied  by  an  enormous  but  painless  inflammatory  swelling  which 
may  extend  throughout  the  cellular  tissue  of  the  trunk.  Most  frequently 
the  disease  is  fatal,  but  if  the  animal  recover  it  will  be  immune  both  to  inocu- 
lation and  to  the  spontaneous  disease.  If  the  animal  die,  the  meshes  of  the 
connective  tissue  are  found,  post  mortem,  to  be  distended  with  a  clear  serous 
fluid  which  is  present  in  such  large  amount  that  occasionally  several  litres 
can  be  collected.  The  cedema  never  affects  the  lungs  or  internal  organs  : 
the  animal  dies  of  an  intoxication. 

(ii)  If,  on  the  other  hand,  the  inoculation  be  made  into  the  dense  cellular 
tissue  at  the  tip  of  the  tail,  the  disease  is  usually  benign  ;  the  swelling  at  the 
site  of  inoculation  is  very  little  marked  and  not  extensive,  and  in  the  great 
majority  of  cases  the  illness  which  follows  is  slight  and  the  animal  quickly 
recovers  ;  it  is  then  found  to  be  immune. 


PLEURO-PNEUMONIA  837 

Vaccination. — Willens  applying  these  observations  vaccinated  animals 
against  pleuro-pneumonia  by  inoculating  a  drop  of  the  pulmonary  exudate 
into  the  cellular  tissue  of  the  tail.  As  the  exudate  quickly  loses  its  virulence 
a  practical  difficulty  was  at  first  experienced,  for  the  exudate  had  to  be 
collected  from  a  recently  killed  animal ;  but  after  Pasteur  had  demonstrated 
that  the  exudate  when  collected  with  aseptic  precautions  will  retain  its  pro- 
perties for  several  (up  to  4)  weeks  vaccination  became  an  easier  matter.  If 
the  exudate  be  inoculated  4-6  weeks  after  collection  it  produces  no  effect ; 
hence  the  necessity,  in  countries  where  prophylactic  vaccination  is  a  regular 
practice,  of  having  vaccination  centres  where  calves  can  be  inoculated  and 
the  fresh  material  collected 'at  least  once  a  month  (but  vaccination  is  now 
effected  with  pure  cultures  (vide  infra}). 

2.  Methods  of  diagnosing-  the  disease— Characteristics 
of  the  organism. 

The  microscope  and  the  ordinary  methods  of  cultivation  being  of  no  use 
in  searching  for  the  micro-organism  in  pleuro-pneumonia,  Nocard  and  Roux 
cultivated  the  exudate  in  collodion  sacs. 

Collodion  sacs  (p.  175)  are  filled  with  broth,  sown  with  a  drop  of  the 
exudate  from  a  case  of  pleuro-pneumonia  and  introduced  into  the  peritoneal 
cavities  of  rabbits.  After  remaining  there  for  a  fortnight  or  3  weeks  their 
contents  are  cloudy,  opalescent,  and  slightly  albuminous.1 

Microscopical  examination  of  this  cloudy  fluid  with  a  magnification  of  about 
2000  diameters  shows  that  it  contains  a  large  number  of  motile  refractile 
points  but  so  small  that  their  shape  cannot  be  made  out  and  they  cannot 
be  stained. 

The  virus  is  not  without  effect  upon  the  rabbits  used  for  the  experiments  :  when 
the  sacs  are  removed  after  15-20  days  the  animals  are  found  to  be  very  thin,  and 
they  occasionally  die  before  the  twentieth  day  in  a  state  of  extreme  emaciation 
but  without  any  appreciable  lesion :  their  organs  and  body-fluids  are  sterile.  The 
control  animals  in  which  similar  but  sterile  sacs  are  inserted  remain  healthy.  The 
symptoms  are  evidently  of  the  nature  of  a  toxaemia,  which  must  be  due  to  the  diffusion 
of  products  elaborated  by  the  micro-organism  :  and  the  experiment  shows  that  the 
rabbit  is  susceptible  to  the  toxin  though  it  is  immune  to  the  organism  itself. 

The  contents  of  the  collodion  sacs  removed  from  the  rabbits  cannot  be 
cultivated  on  ordinary  culture  media.  The  vitality  of  the  virus  can  be 
preserved  by  keeping  it  in  collodion  sacs  in  the  peritoneal  cavities  of  these 
animals,  but  its  virulence  appears  to  diminish  in  the  process. 

The  organism  cannot  be  grown  in  collodion  sacs  in  the  peritoneal  cavities 
of  guinea-pigs. 

After  a  long  series  of  experiments,  Nocard  and  Roux  have  succeeded  in 
devising  an  artificial  medium  on  which  the  micro-organism  of  pleuro-pneu- 
monia can  be  grown  in  vitro.  The  medium  is  a  mixture  of  twenty  parts  of 
Martin's  peptone  solution  (p.  32)  and  one  part  of  rabbit  or  cow  serum. 
Tubes  of  this  mixture  sown  aerobically  with  a  drop  of  the  exudate  from  a 
case  of  pleuro-pneumonia  or  with  some  of  the  contents  of  a  collodion-sac 
culture,  and  incubated  at  37°  C.  give  a  growth  similar  to  that  obtained  in 
collodion  sacs  :  moreover  the  micro-organism  retains  its  virulence  on  this 
medium  and  a  long  series  of  sub-cultures  can  be  made. 

By  adding  agar  to  the  above  solution  a  solid  medium  can  be  obtained  on 
which  after  incubating  for  3  or  4  days  the  organism  of  pleuro-pneumonia 
gives  very  minute  colonies.  When  the  colonies  are  very  closely  packed 

1  The  contents  of  control  sacs,  prepared  in  the  same  manner  but  not  sown  with  the 
exudate  remain  clear  under  similar  conditions. 


838  THE   FILTRABLE   VIRUSES 

together  they  form  a  hardly  visible  roughening  of  the  surface  of  the  agar  : 
when  sufficiently  far  apart  they  may  attain  the  size  of  a  pin's  head.  These 
colonies  can  be  stained  en  masse  and  if  they  be  transferred  intact  to  a  slide, 
preparations  can  be  obtained  which  stain  with  the  ordinary  aniline  dyes  and 
are  gram-positive. 

The  inoculation  of. cows  with  cultures  obtained  by  Nocard  and  Roux's 
method  is  followed  by  a  typical  attack  of  the  experimental  disease  :  occa- 
sionally the  animal  dies  :  when  it  recovers  it  is  immune  to  inoculation  with 
cultures  or  with  the  exudate  from  a  case  of  pleuro-pneumonia.  Pure  cultures 
are  now  used  in  place  of  the  serous  exudate  for  the  purpose  of  Willens'  vaccina- 
tion (vide  ante}. 

Filtration. — If  the  exudate  from  a  case  of  pleuro-pneumonia  be  filtered 
through  a  Chamberland  (F)  or  Berkefeld  bougie,  the  filtrate  does  not  produce 
the  disease  in  calves  neither  does  it  give  cultures  :  but,  on  the  other  hand, 
if  the  same  exudate  be  diluted  with  20-30  volumes  of  water  the  filtrate  is 
infective  and  grows  on  serum-broth  ;  under  these  conditions  the  organisms 
pass  through  the  filters  (save  Ohamberland  B  through  which  they  never 


Filtration  thus  enables  a  pure  culture  of  the  organism  to  be  obtained  from 
contaminated  exudates,  ordinary  bacteria  being  held  back  by  the  filter. 

SECTION  II.— THE  VIRUS   OF  FOOT  AND   MOUTH  DISEASE. 

(Aphthous  fever.) 

Foot  and  mouth  disease  infects  cattle,  sheep,  goats,  and  pigs  and  is  trans- 
missible to  man. 

Microscopical  examination  and  cultural  methods  fail  to  reveal  the  presence 
of  any  micro-organism  in  the  lymph  contained  in  unbroken  vesicles,  but  this 
lymph  is  nevertheless  infective  and  on  inoculation  into  cattle,  pigs,  sheep  and 
goats  will  reproduce  the  disease. 

Lceffler  and  Frosch  have  shown  that  the  virus  of  foot  and  mouth  disease 
is  an  invisible  organism,  which  easily  passes  through  a  Berkefeld  bougie  if 
care  be  taken  to  dilute  the  serous  fluid  with  40-50  volumes  of  water  but 
is  held  back  by  closer  filters  such  as  Kitasato's  bougie  and  Chamberland  B. 

Vaccination.  Serum  therapy.— Aphthous  lymph  loses  its  infectivity  if 
kept  for  some  time  (3-8  weeks),  or  if  heated  (12  hours  at  37°  C.  or  30 
minutes  at  60°-70°  C.).  By  inoculating  into  the  veins  of  an  ox  a  mixture 
of  an  old  inactive  lymph  and  fresh  lymph  attenuated  by  heating  at  60°  C. 
for  5  minutes  Loeffler  has  been  able  to  produce  an  immunity  to  the  disease, 
but  this  immunity  is  only  acquired  slowly.  By  hyper-immunizing  oxen  with 
increasing  doses  of  an  active  lymph  of  constant  virulence,  Loeifler  obtained 
a  serum  which  has  some  slight  prophylactic  properties. 

When  a  stable  is  found  to  be  infected  and  infection  of  all  the  animals 
in  it  has  become  inevitable,  recourse  may  be  had  to  an  emergency  inocula- 
tion :  all  the  animals  are  purposely  infected  in  such  a  manner  as  to  secure 
that  they  shall  suffer  from  the  least  severe  form  of  the  disease.  For  this 
purpose  the  tongues  and  the  inner  surface  of  the  lips  of  the  healthy  animals 
are  rubbed  with  a  rough  cloth  soaked  in  virulent  saliva. 

SECTION  III.— THE  VIRUS   OF  HORSE-SICKNESS. 

Horse  sickness  is  a  fatal  disease  affecting  horses  in  South  Africa  which  can 
be  experimentally  inoculated  and  though  not  spontaneously  contagious  is 
apparently  transmitted  by  biting  insects. 


HORSE-SICKNESS  839 

The  blood  and  the  pulmonary  and  conjunctival  exudates  are  infective. 
No  micro-organisms  have  been  demonstrated  in  these  fluids  neither  have 
organisms  been  cultivated  from  them. 

The  filtrate  obtained  by  filtering  the  undiluted  exudate  or  blood  mixed 
with  serum  through  a  Berkefeld  or  Chamberland  F  bougie  will  reproduce 
the  disease  on  inoculation  into  a  susceptible  animal  (McFadyean).  The  virus 
will  pass  through  a  Chamberland  B  bougie  provided  that  the  exudate  has  been 
diluted  with  30  volumes  of  water. 

In  places  where  horse-sickness  is  prevalent  a  very  similar  disease  is  observed  in 
sheep,  goats  and  cows.  This  latter  disease  is  known  as  catarrhal  fever  of  sheep  (blue 
tongue,  mouth  sickness,  heart- water,  catarrhal  malarial  fever,  etc.) :  it  appears  to 
be  different  from  horse- sickness,  and  is  also  caused  by  a  "  filter  passer."  The 
filtrate  obtained  by  filtering  the  blood  or  serum  (Berkefeld  bougie)  is  infective 
(Theiler  and  Robertson). 


SECTION  IV.— THE  VIRUS   OF  RINDERPEST. 

(Cattle  plague.) 

Rinderpest  is  essentially  a  disease  of  bovine  animals,  but  it  also  affects 
some  races  of  sheep,  goats  and  pigs.  The  blood,  exudates  and  juices  from 
the  internal  organs  are  infective,  but  the  causal  micro-organism  can  neither 
be  seen  nor  cultivated.  The  virus  easily  passes  through  a  Berkefeld  bougie 
and,  under  certain  conditions  and  with  difficulty,  through  a  Chamberland 
F  bougie. 

SECTION  V.— THE  VIRUS   OF  BIRD  PLAGUE. 

Bird  plague  must  be  carefully  distinguished  from  fowl-cholera  (Centanni 
and  Savonuzzi).  The  disease,  which  is  fairly  widely  distributed,  may  affect 
all  farm-yard  birds  and  especially  pheasants  ;  it  is  due  to  an  invisible  organism 
(Maggiora  and  Valenti).  The  pleural,  pericardial  and  peritoneal  exudates  and 
the  blood  of  diseased  birds  are  infective.  The  virus  will  pass  through  a 
Berkefeld  or  Chamberland  F  bougie  and  even  through  Chamberland  B. 

Marchoux  has  succeeded  in  growing  the  virus  of  bird  plague  :  after  ten 
successive  sub-cultures,  a  dose  of  0*20  c.c.  of  the  blood  in  which  the  virus 
was  sown  killed  fowls  in  2  days.  The  cultures  are  grown  at  37°  C.  in 
defibrinated  fowl  blood  spread  on  a  thick  layer  of  glucose-peptone-agar : 
growth  does  not  take  place  through  the  whole  of  the  blood  but  only  in  a 
.zone  near  the  surface  of  the  agar. 


SECTION  VI.— THE  VIRUS  OF  SHEEP-POX. 

Synonym. —  Variola  ovina  ;  Fr.  La  Clavelee. 

The  virus  is  present  in  the  pustules  and  in  all  the  lesions  of  sheep-pox. 
No  organism  has  been  discovered.  The  virus  cannot  be  cultivated  and 
does  not  remain  in  the  blood. 

The  juice  obtained  by  scraping  the  pustules  when  diluted  with  water  and 
filtered  through  a  Berkefeld  bougie  will  yield  an  infective  filtrate.  The  virus 
will  not  pass  through  a  Chamberland  F  bougie  if  the  material  be  filtered 
rapidly  and  at  once,  but  if  the  filtration  be  carried  on  continuously  for  from 
1-7  days  the  virus  will  then  appear  in  the  filtrate  (Borrel). 

When  the  virus  is  diluted  with  non-sterilized  tap  water  a  number  of  very  small 
vibrios  and  spirilla  pass  through  the  bougie  with  the  virus  of  sheep-pox  :  these 
water  organisms  (which  can  be  stained  by  Lceffler's  method,  p.  149)  multiply  in 


840  THE  FILTRABLE   VIRUSES 

the  filtrate  when  it  is  kept  at  20°  C.,  but  will  not  grow  in  ordinary  broth.  The  filtrate 
as  the  result  of  the  multiplication  of  these  organisms  becomes  slightly  opalescent. 
Under  similar  conditions,  the  filtrate  occasionally  contains  certain  structures  which 
Borrel  regards  as  belonging  to  the  Protozoa  and  to  which  he  has  given  the  name 
Micromonas  mesnili. 

With  the  object  of  protecting  sheep  against  sheep-pox  the  animals  are 
immunized  by  infecting  them  with  a  mild  form  of  the  disease.  Vaccination 
[or  clavelization]  is  effected  by  inoculating  a  small  quantity  of  lymph  from 
the  pustules  with  a  lancet  on  the  tail  or  on  the  internal  surface  of  the  ear. 

The  inoculation  is  not  without  danger  :  some  of  the  animals  die  (1-10  per 
cent.).  Borrel  hyper-immunized  sheep  which  had  recovered  from  sheep- 
pox  by  inoculating  them  on  several  occasions  with  lymph  from  the  inoculation 
pustule,  and  obtained  a  serum  which  had  prophylactic  and  therapeutic 
properties  :  the  inoculation  of  10-20  c.c.  of  this  serum  has  arrested  the 
mortality  in  flocks  exposed  to  infection. 

"Infectious  epithelioses." 

Borrel  has  introduced  the  term  "  infectious  epithelioses  "  to  denote  a  number  of 
diseases  having  a  special  affinity  for  the  epithelial  tissues  and  caused  by  "  filter- 
passing  organisms "  :  sheep-pox,  cow-pox,  foot  and  mouth  disease,  rinderpest, 
epithelioma  contagiosum  of  fowls  and  molluscum  contagiosum.  In  sheep-pox  there  is 
always  present  a  characteristic  and  specific  element,  the  sheep-pox  cell,  having  a 
vacuolated  nucleus  with  pseudo-parasitic  inclusions  (due  probably  to  the  penetra- 
tion of  poly-morpho-nuclear  cells  which  in  these  cells  undergo  a  process  of  degenera- 
tion) .  wherever  the  virus  settles  it  produces  a  proliferation  of  the  epithelial  tissues, 
and  (in  the  liver,  kidney  and  lung)  epithelial  growths  which  develop  at  the  expense 
of  the  pre-existing  cells  of  the  part.  There  can  be  no  doubt  but  that  these  changes 
bear  a  considerable  resemblance  to  the  evolution  of  cancer  growths,  so  that  the 
hypothesis  might  be  put  forward  that  the  cancer  virus  enters  into  the  category  of 
filter-passing  organisms.  This  is  merely  an  hypothesis,  and  it  must  be  noted,  as 
Borrel  says,  that  the  metastases  of  sheep- pox  are  absolutely  different  from  cancer 
metastases  :  for  example,  the  metastases  in  the  lung  in  the  former  case  represent  a 
proliferation  of  pre-existing  cells,  while  cancer  metastases  are  produced  by  a  graft 
in  the  lung  of  cancer  cells  from  the  original  tumour. 


SECTION  VII.— THE  VIRUS  OF  COW  POX. 

[Variola  vaccinia.] 

If  the  fresh  exudate  from  the  vesicles  of  an  heifer  suffering  from  cow  pox 
be  rubbed  up  with  10-12  times  its  weight  of  water  and  filtered  through  a 
Berkefeld  V  bougie  a  filtrate  is  obtained  which  according  to  many  investigators 
has  proved  to  be  infective. 

The  virulence  is  only  manifested  if  before  filtration  the  lymph  is  left  to 
macerate  for  a  long  time  in  sterile  water  (Carini}  Negri).  The  first  emulsion 
of  lymph  is  left  in  the  ice  chest  for  2  or  3  days  then  rubbed  up  and  replaced 
in  the  ice  chest  for  a  fortnight.  It  is  not  until  now  that  the  product  is  filtered, 
first  through  wool  then  through  paper  and  finally  through  a  Berkefeld  bougie. 

Negri  soaked  up  the  filtrate  on  a  small  piece  of  sterile  absorbent  wool  and  placed 
it  on  the  cornea  of  a  rabbit  (which  had  been  previously  scarified)  for  about  10  hours. 
A  typical  pustule  developed  the  contents  of  which  were  infective  and  produced 
similar  effects  on  the  cornese  of  other  rabbits  in  series,  and  on  the  skin  of  a  calf. 
With  a  similar  filtrate  Remlinger  and  Osman  Nouri  have  been  able  to  produce  a 
typical  vaccinal  eruption  on  the  shaved  skin  of  guinea-pigs  and  rabbits. 

In  these  investigations,  the  inoculations  should  be  performed  on  a  number  of 
animals :  for  the  virus  is  partially  retained  by  the  bougie,  and  the  filtrate  is  conse- 
quently not  highly  infective  (p.  836). 

The  filtrate  inoculated  beneath  the  skin  of  a  susceptible  animal  immunizes 


RABIES  841 

the  latter  against  vaccine  lymph.  This  fact,  which  was  first  shown  by 
Casagrandi  and  has  also  been  observed  by  Rouget,  and  by  Remlinger  and 
Osman  Nouri,  again  demonstrates  that  the  virus  will  pass  through  porcelain 
or  similar  filters. 

Rouget  experimented  on  ten  heifers,  by  inoculating  40  c.c.  of  the  filtrate  beneath 
the  skin  :  the  test  inoculation  was  carried  out  a  week  later  with  glycerin  lymph 
with  which  controls  were  also  inoculated.  He  obtained  four  positive  results. 

SECTION  VIII.— THE  VIRUS   OF  YELLOW  FEVER. 

In  yellow  fever  the  blood  is  infective,  and  if  inoculated  into  an  healthy 
man  leads  to  the  development  of  the  disease  (Reed,  Carroll,  Agramonte). 
The  disease  is  transmitted  by  a  mosquito  (Stegomyia  fasciata)  :  after  feeding 
on  the  blood  of  a  yellow  fever  patient  this  mosquito  can  infect  a  healthy 
man. 

Reed,  Carroll  and  Agramonte  have  shown  that  yellow  fever  is  due  to  an 
invisible  micro-organism.  From  their  experiments,  which  have  been  con- 
firmed by  those  of  Parker,  Beyer  and  Pothier,  Rosenau,  Marchoux,  Salimbeni 
and  Simond,  it  follows  that  serum  or  defibrinated  blood  from  a  case  of  yellow 
fever  diluted  with  an  equal  volume  of  water  and  filtered  through  a  Berkefeld 
or  Chamberland  F  or  B  bougie  will  yield  an  infective  filtrate.  The  virus 
seems  to  pass  through  filters  quite  easily  and  in  most  cases  a  dose  of  the 
filtrate  corresponding  to  O5-1  c.c.  of  serum  has  been  sufficient  to  cause 
typical  yellow  fever  in  man. 

SECTION  IX.— THE  VIRUS   OF  RABIES. 

(Hydrophobia.) 

Remlinger  and  Riffat-Bey  have  shown  that  the  virus  of  rabies  will  readily 
pass  through  a  Berkefeld  V  and  even  a  W  or  N  and  Chamberland  F :  their 
experiments  were  carried  out  with  the  brain  of  a  rabbit  made  into  an  emulsion 
with  300  c.c.  of  water.  Similar  results  have  been  obtained  by  di  Vestea, 
Schiider,  Bertarelli  and  Volpino,  de  Blasi  and  Celli. 

Peculiar  polychromatic  structures  never  seen  in  normal  tissues  have  been 
described  by  Negri  in  the  central  nervous  system  of  man  and  the'  lower 
animals  which  have  succumbed  to  rabies. 

These  structures,  which  are  invariably  intra-cellular,  occur  in  the  pyramidal  cells 
of  the  cornu  ammonis,  in  the  cells  of  Purkinje,  in  the  cerebellum  and  in  the  large 
cells  of  the  cerebral  convolutions  :  they  are  not  found,  or  only  very  rarely  indeed, 
in  the  cells  of  the  pons  varolii  and  medulla  oblongata.  They  are  generally  round  or 
oval  and  measure  from  10-25/x  in  diameter:  occasionally  they  are  much  smaller 
and  only  measure  0.5-1/x  or  less.  They  stain  an  intense  bright  red  by  Fasoti'a 
method. 

Fasoti's  method. — 1.  Fix  small  pieces  of  the  tissue  for  24-48  hours  in  Foa's  solu- 
tion (Miiller's  solution,  100  c.c.  ;  perchloride  of  mercury,  2  grams)  or  in  acid  per- 
chloride  (p.  189). 

2.  Wash  rapidly  in  water.     Freeze  and  cut.     If  a  precipitate  be  produced  it  can 
be  removed  by  washing  in  iodine- alcohol. 

3.  Stain  the  sections  for  5-10  minutes  in  0'5  per  cent,  solution  of  eosin,  heating 
gently.     Wash  in  water. 

4.  Differentiate  until  +he  sections  acquire  a  pink  tint  in  the  following  solution : 

1  per  cent,  aqueous  solution  of  caustic  soda,       -  4  drops. 

90  per  cent,  alcohol,         -  -.50  c.c. 

Wash  in  water. 

5.  Stain  in  a  0*25  per  cent,  aqueous  solution  of  methylene  blue  until  the  sections 
are  pale  violet  in  colour. 


842  THE   FILTRABLE   VIRUSES 

6.  Wash  for  1  or  2  minutes  in  50  per  cent,  alcohol,  pass  rapidly  through  absolute 
alcohol,  and  xylol.  Mount  in  balsam. 

Celli,  and  de  Blasi,  and  others  regard  the  Negri  bodies  as  parasites  which  at 
a,  certain  stage  in  their  life  history  are  so  small  as  to  be  capable  of  passing 
through  niters,  and  consider  that  it  is  these  minute  forms  which  originate 
the  infection.  Remlinger  holds  that  the  Negri  bodies  are  merely  changes  in 
the  nerve  cells  following  the  infection  of  the  latter  by  the  ultra-microscopic 
parasite  of  the  disease. 


SECTION  X.— FILTRABLE  VIRUSES   IN  THE   PASTEURELLOSES. 

1.  Distemper. — Carre  by  filtering  the   nasal  discharge   of  dogs  infected 
with  distemper  obtained  a  liquid  which  though  apparently  sterile  produced 
all  the  symptoms  of  distemper  when  inoculated  into  young  dogs.     [M'Gowan 
has  isolated  a  gram-negative  bacillus  from  the  respiratory  passages  of  animals 
suffering  from  "  distemper  "  and  brings  forward  evidence  to  show  that  this 
organism  is  the  cause  of  the  disease  (p.  459)]. 

2.  Infectious  anaemia  of  horses. — Carre  and  Vallee,  by  filtering  through  a 
special  bougie  rather  more  porous  than  Berkefeld  V  a  mixture  of  one  part  of 
serum  from  a  horse  suffering  from  this  disease  and  four  parts  of  normal 
saline  solution,  obtained  a  virulent  nitrate.     When  this  filtrate  is  inoculated 
into  the  jugular  vein  of  an  horse  in  doses  of  500  c.c.  it  produces,  after  an 
incubation  period  of  6  days,  an  anaemia  which  runs  a  characteristic  course 
and  which  can  be  transmitted  from  one  animal  to  another.     The  virus  will 
also  pass  through  a  Berkefeld  V  or  Chamberland  F  or  B  but  the  incubation 
period  under  these  circumstances  is  of  longer  duration. 

3.  Bird  diphtheria. — ^tiologically  bird   diphtheria  is  a  totally  different 
disease  from  human  diphtheria.     Guerin  thought  it  was  due  to  a  cocco- 
bacillus  belonging  to  the  Pasteurella  group  which  he  found  in  the  heart 
blood  of  infected  birds  :    this  organism  on  inoculation  however  gave  rise  to 
a  fatal  septicaemia  quite  different  from  the  naturally  acquired  disease. 

By  grinding  up  in  normal  saline  solution  the  nictitating  membrane  of  a 
fowl  which  had  been  infected  with  a  thread  dipped  in  an  emulsion  of  a  false 
membrane,  Bordet  obtained  an  emulsion  which  produced  in  fowls  the  typical 
false  membranes  of  bird  diphtheria.  When  this  emulsion  was  sown  on 
blood  agar,  the  only  visible  growth  consisted  of  a  few  colonies  of  adventitious 
organisms  :  but  by  scraping  the  agar  where  there  was  no  visible  growth  with 
a  platinum  wire,  and  transferring  the  scrapings  to  a  little  drop  of  water  and 
rubbing  the  mucous  membrane  of  the  mouth  with  the  emulsion,  false  mem- 
branes were  produced  in  a  normal  fowl.  Serial  cultures  can  also  be  obtained 
and  occasionally  extremely  small  colonies  are  visible.  Under  the  micro- 
scope an  emulsion  of  the  cultures  shows  very  large  numbers  of  small  granular 
dots  generally  collected  together  in  masses.  This  organism  and  that  of 
pleuro-pneumonia  seem  to  be  the  smallest  yet  cultivated. 

According  to  Carnwath,  this  filtrable  virus  appears  to  be  identical  with 
that  of  molluscum  contagiosum  of  birds  (vide  ante).  In  an  epizootic  of 
diphtheria  among  birds  investigated  by  him  the  virus  produced  indifferently 
molluscum  contagiosum,  or  diphtheria  according  as  to  whether  the  material 
was  inoculated  on  the  bucco-pharyngeal  mucous  membrane  or  on  the  comb. 

[G.  Dean  and  Marshall  have  recorded  an  outbreak  of  diphtheria  in  the 
wood  pigeon  apparently  due  to  a  filtrable  virus.  By  painting  a  filtered 
(Berkefeld  filter)  emulsion  of  a  membrane  from  an  infected  bird  on  to  the 
throat  of  doves  they  were  able  to  reproduce  the  disease  experimentally.] 


SWINE   FEVER  '843 

SECTION   XL— THE  VIRUS   OF   SWINE  FEVER   OR   HOG-CHOLERA. 

The  role  which  a  filter-passing  virus  is  believed  to  play,  according  to  recent 
research,  in  the  aetiology  of  hog  cholera  has  already  been  adverted  to  when 
discussing  the  bacillus  known  as  Bacillus  aertrycke  (p.  438).  [The  conclusion 
is  that  Hog-cholera  or  swine  fever  is  due  to  a  filtrable  virus  present  in  the 
blood  of  the  sick  animals  and  that  the  hog  cholera  bacillus  (bacillus  cholera; 
suis  of  Salmon  and  Theobald  Smith)  is  a  secondary  infection,  which  is  com- 
monly present,  and  which  may  increase  the  mortality  among  infected  animals. 

[These  facts  established  first  by  Dorset  and  M'Bryde  in  America  have  been  fully 
confirmed  by  MTadyean  and  Stockman  in  England,  by  Uhlenhuth  in  Germany 
and  by  many  other  observers  in  countries  where  swine  fever  is  prevalent. 

[The  virus  of  swine  fever  will  pass  through  a  Chamberland  F  porcelain 
bougie  :  if  the  blood  of  a  sick  animal  be  diluted  ten  times  it  will,  after  filtra- 
tion through  this  bougie,  on  inoculation  in  suitable  dose  into  a  young  pig 
(10-20  c.c.)  give  rise  to  a  typical  attack  of  swine  fever ;  and  healthy  pigs 
kept  in  contact  with  an  infected  animal  will  contract  the  disease  (Stockman). 

[Pigs  are  the  only  susceptible  animals  and  after  recovery  from  the  disease 
are  immune  to  further  infection.  This  natural  immunity  after  a  natural 
attack  of  the  disease  is  an  important  fact  in  establishing  that  the  filtrable 
virus  and  not  the  hog  cholera  bacillus  is  the  cause  of  the  disease.  Animals 
immunized  against  this  bacillus  are  not  immune  to  swine  fever. 

[The  virus  is  present  in  the  blood  and  in  all  the  internal  organs  of  animals  suffer- 
ing from  swine  fever.  It  is  also  found  in  the  urine,  secretions  from  the  eyes  and 
nose  and  in  the  pustular  eruption  on  the  skin  (Uhlenhuth).  The  excreta  do  not 
appear  to  be  infective — or  to  be  more  precise,  are  not  an  important  source  of 
infection.  Some  animals  appear  to  retain  the  virus  for  some  time  after  the  acute 
symptoms  have  subsided. 

[Healthy  animals  can  be  infected  through  the  mouth,  skin  or  the  mucous  mem- 
branes. The  disease  can  also  be  transmitted  by  inhalation. 

[The  virus  is  highly  resistant  to  external  influences.  It  can  be  preserved 
in  animal  tissues  or  fluids  for  many  months  either  at  room  temperature 
or  in  the  ice  chest.  Desiccation  appears  to  have  no  effect  upon  the  virulence 
of  infected  material,  nor  does  heating  at  58°  C.  for  2  hours  :  but  heating  at 
72°  C.  for  1  hour  destroys  the  virus.  The  virus  is  more  resistant  to  the 
action  of  antiformin  than  the  hog-cholera  bacillus. 

[Certain  cell  inclusions  are  found  in  smear  preparations  from  the  conjunctivae  of 
almost  every  animal  suffering  from  swine  fever.  These  are  similar  to  the  cell  inclu- 
sions (Chlamydozoa)  described  by  Prowazek  and  Halberstadter  in  trachoma  which 
they  were  inclined  to  regard  as  the  cause  of  this  disease.  No  evidence  has  yet  been 
adduced  in  favour  of  the  parasitic  nature  of  these  cell  inclusions  (cf.  Negri  bodies 
in  Rabies). 

Artificial  immunization. — [Starting  from  the  well  established  fact  that  pigs 
which  have  recovered  from  swine  fever  are  immune  to  the  disease  attempts 
have  been  made  to  induce  an  artificial  immunity  by  inoculating  susceptible 
animals  with  the  serum  of  hyper-immunized  animals. 

[Pigs  which  have  recovered  from  an  attack  of  the  naturally-acquired 
disease  are  hyper-immunized  by  inoculating  them  sub-cutaneously,  intra- 
venously or  intra-abdominally  with  the  filtered  serum  or  defibrinated  blood 
of  infected  animals. 

[The  serum  is  prophylactic  rather  than  curative,  and  therefore  if  it  is  to 
be  utilized  to  the  greatest  advantage  in  an  outbreak  it  should  be  used  at 
the  earliest  possible  moment  after  the  appearance  of  the  disease.  A  dose 
of  about  20  c.c.  should  be  inoculated  sub-cutaneously  as  a  prophylactic 
measure. 


844  THE   FILTRABLE   VIRUSES 

[In  America  and  on  the  Continent  the  results  appear  to  be  most  satisfactory. 
When  used  at  an  early  stage  of  an  outbreak  2*9  per  cent,  of  the  treated  animals 
died  against  93  per  cent,  of  the  untreated  (Uhlenhuth)  :  in  herds  where  hog- 
cholera  existed  13  per  cent,  of  the  treated  died  against  75  per  cent,  of  the 
untreated  :  in  herds  which  had  been  exposed  to  disease  4  per  cent,  of  the 
treated  died  against  89  per  cent,  of  the  untreated  (Dorset).] 


SECTION  XII.— THE  VIRUS   OF  ACUTE  ANTERIOR  POLIOMYELITIS.1 

Syn. — Infantile  paralysis.     Fr.  La  poliomyelite  epidemique  :   Maladie  de 

Heine- Medin. 

Landsteiner  and  Popper  in  1908  were  the  first  to  show  that  acute  anterior 
poliomyelitis  could  be  reproduced  in  monkeys  by  inoculating  into  the  peri- 
toneal cavity  an  emulsion  of  the  spinal  cord  of  an  affected  individual.  Leva- 
diti  and  Landsteiner  further  demonstrated  that  the  effect  produced  in  the 
monkey  was  not  simply  the  result  of  the  inoculation  of  a  toxin  but  was  a 
true  infection.  These  observations  were  soon  confirmed  by  Flexner  and 
Lewis  in  New  York,  Leiner  and  Wiesner  in  Vienna  and  subsequently  by  many 
other  observers. 

The  virus  of  acute  anterior  poliomyelitis  belongs  to  the  group  of  filtrable  viruses  : 
if  pieces  of  the  spinal  cord  of  a  child  who  has  died  of  the  disease  be  emulsified  in 
normal  saline  solution  and  the  emulsion  be  filtered  through  a  Chamberland,  Berke- 
feld  or  Reichel  filter  (in  vacua  under  a  pressure  of  30-40  cm.)  the  filtrate  inoculated 
into  susceptible  animals  will  be  followed  by  the  symptoms  and  lesions  of  the 
disease  ;  moreover  the  virus  can  be  passed  from  animal  to  animal,  a  fact  which 
proves  that  it  is  a  living  proliferating  organism. 

The  organism  has  never  been  seen  neither  have  attempts  to  cultivate  it  succeeded  : 
if  a  virulent  filtrate  be  sown  on  culture  media  the  cultures  remain  sterile,  and  if  a 
drop  of  the  filtrate  be  examined  microscopically  no  organism  can  be  seen  in  it. 

The  virus  can  be  preserved  unaltered  in  the  ice-chest  in  a  glycerin-saline  solution 
(1  to  2)  for  considerable  periods  of  time  (5  months,  Rcemer  and  Joseph)  and  in 
this  respect  resembles  the  viruses  of  rabies  and  variola  vaccinia.  Emulsions  of 
virulent  cords  will  retain  their  infectivity  for  at  least  a  fortnight  when  dried  in 
vacuo  over  sulphuric  acid  (Levaditi  and  Landsteiner).  Similarly  portions  of  the 
spinal  cord  placed  in  bottles  over  potash  and  kept  in  the  dark  are  found  to  be  infective 
after  the  lapse  of  24  days. 

The  organism  of  acute  anterior  poliomyelitis  is  localized  chiefly  in  the  spinal  cord, 
medulla  oblongata  and  intervertebral  ganglia  but  has  been  found  in  the  cervical 
cortex  (Flexner  and  Lewis)  and  in  the  olfactory  bulbs  (Levaditi  and  Landsteiner, 
Flexner).  It  does  not  under  ordinary  circumstances  remain  long  hi  the  cerebro- 
spinal  fluid  or  in  the  blood-stream. 

Experimental  infection. 

Apes  and  monkeys  are  practically  the  only  animals  susceptible  to  infection 
with  the  virus  of  acute  anterior  poliomyelitis  ;  most  laboratory  animals 
appear  to  be  immune,  though  in  some  cases  atypical  symptoms  and  lesions 
have  been  produced  in  rabbits. 

Methods  of  infection. — Monkeys  can  be  experimentally  infected  by  almost 
any  method  of  inoculation  :  intra-peritoneal,  intra-cerebral,  intra-ocular, 
intra-venous,  by  the  nasal  mucous  membrane,  etc.  The  disease  can  also  be 
set  up  in  these  animals  by  feeding  them  upon  infected  material  (Leiner  and 
Wiesner).  It  is  to  be  noted  however  that  in  Levaditi  and  Landsteiner 's 
experience  the  disease  cannot  be  produced  by  rubbing  the  virus  into  the 
scarified  surface  of  the  skin  and  that  sub-cutaneous  inoculation  cannot  be 

1  This  section  has  been  added. 


ACUTE   ANTERIOR   POLIOMYELITIS  845 

relied  upon  to  infect  the  animal.  Infection  follows  if  the  virus  be  inoculated 
into  the  sheath  of  a  nerve  trunk  ;  this  is  of  interest  in  that  it  shows  that  the 
virus  travels  up  the  nerves,  probably  along  the  lymphatics. 

Further,  after  inoculation  of  the  virus  into  a  nerve  trunk  the  characteristic 
paralyses  always  appear  first  in  the  limb  supplied  by  that  nerve  (Flexner 
and  Lewis,  Levaditi  and  Landsteiner,  Leiner  and  Wiesner). 

With  regard  to  infection  by  the  respiratory  passages  :  In  their  earlier 
experiments  Levaditi  and  Landsteiner  failed  to  infect  monkeys  either  by 
painting  the  mucous  membranes  of  the  nose  and  back  of  the  throat  with  an 
emulsion  of  the  virus  or  by  plugging  the  nasal  fossae  with  wool  soaked  in  the 
virus,  but  succeeded  when  they  inoculated  the  infected  material  beneath  the 
nasal  mucous  membrane.  Flexner  and  Lewis  also  infected  animals  by 
painting  the  virus  on  the  scarified  surface  of  the  nasal  and  pharyngeal 
mucous  membranes. 

Leiner  and  Wiesner,  on  the  other  hand,  were  able  to  infect  monkeys  by 
painting  the  nasal  mucous  membrane  without  previous  scarification,  by 
inhalation  and  by  injecting  the  virus  into  the  trachea  and  though  Levaditi 
and  Landsteiner  suggested  as  a  possible  explanation  of  these  results  that  the 
monkeys  used  by  Leiner  and  Wiesner  may  have  had  at  the  time  of  the 
experiment  some  small  abrasions  on  the  mucous  membranes  which  escaped 
their  observation,  it  is  now  established  that  the  virus  passes  with  readiness 
and  constancy  from  the  intact,  or  practically  intact,  mucous  membrane  of 
the  nose  to  the  central  nervous  system  (Flexner). 

Experimental  monkeys  have  never  been  observed  to  contract  the  disease 
by  contagion  (Flexner  and  Lewis,  Levaditi  and  Landsteiner,  Leiner  and 
Wiesner,  Roemer). 

Symptoms  generally  appear  after  an  incubation  period  of  about  8  days 
(7-11  days).  The  period  of  incubation  is  rather  longer  when  a  filtered  virus 
is  used  than  when  an  unfiltered  emulsion  is  inoculated  and  also  when  a 
relatively  small  dose  is  inoculated. 

Symptoms. — After  the  period  of  incubation  has  elapsed  certain  prodromal 
symptoms  of  which  the  most  constant  are  twitchings  all  over  the  body  mark 
the  onset  of  the  disease.  These  initial  symptoms  are  followed,  as  a  rule 
within  a  few  hours,  first  by  paralysis  of  the  hind  limbs,  then  of  the  posterior 
part  of  the  body,  trunk,  arms  and  neck  :  the  bulbar  centres  are  subsequently 
affected,  and  death  takes  place  in  2-3  days.  This  is  the  most  usual  course 
for  the  disease  to  run  and  is  known  as  the  Ascendinci  type.  It  resembles 
Landry's  ascending  paralysis  often  observed  during  epidemics  of  acute 
anterior  poliomyelitis. 

In  another  type  known  as  the  superior  type  the  neck  muscles  are  mainly 
involved  and  to  some  extent  the  muscles  of  the  fore  limbs. 

The  experimental  disease  is  occasionally  manifested  by  paralysis  of  the 
motor  cranial  nerves,  generally  the  Vllth  or  the  Illrd. 

In  addition  to  these  types  various  "  mixed  "  paralyses  may  be  produced. 

In  milder  infections  the  acute  symptoms  are  followed  by  paralysis,  mus- 
cular atrophy  and  deformity  :  occasionally  the  animals  recover  completely. 
In  a  few  cases  relapses  occur  and  a  different  part  of  the  body  from  that 
attacked  in  the  first  instance  may  be  involved. 

Immunity. — One  attack  of  the  disease  even  if  atypical  and  abortive  almost 
always  confers  a  complete  and  lasting  immunity. 

The  serum  of  persons  and  of  experimental  animals  which  have  recovered 
from  the  disease  is  microbicidal  in  vitro  (Levaditi  and  Landsteiner,  Flexner 
and  Lewis). 


846  THE   FILTRABLE   VIRUSES 

Prophylactic  vaccination. — Levaditi  and  Landsteiner  by  applying  Pasteur's 
method  of  vaccination  against  rabies  have  successfully  vaccinated  monkeys 
against  acute  anterior  poliomyelitis.  Infected  spinal  cords  are  dried  in 
bottles  over  caustic  potash  for  varying  periods  of  time  (3-9  days)  and  inocula- 
tions of  emulsions  of  the  dried  cords  are  made  at  short  intervals.1 

The  method  is  however  hardly  safe  for  practical  purposes  because  a  cord 
dried  for  24  days  over  caustic  potash  still  contains  the  living  virus  (vide 
supra). 

Flexner  and  Lewis  have  immunized  monkeys  by  inoculating  them  sub- 
cutaneously  with  diluted  emulsions  of  the  virus  gradually  increasing  the 
amount  of  virus  inoculated. 

Though  immune  serums  have  been  proved  to  be  microbicidal  in  vitro  they 
have  no  effect  on  the  course  of  the  disease  when  inoculated  intra-peritoneally 
or  intra-spinally. 

.^Etiology. — Flexner  is  of  opinion  that  in  man  the  nasal  mucous  membrane 
is  the  site  both  of  ingress  and  egress  of  the  virus  of  poliomyelitis  and  from 
experiments  devised  to  determine  the  channel  of  infection  "  the  conclusion 
is  unavoidable  that  the  virus  ascends  by  the  nerves  of  smell  to  the  brain, 
multiplies  in  and  about  the  olfactory  lobes  and  in  time  passes  into  the  cerebro- 
spinal  liquid  which  carries  it  to  all  parts  of  the  nervous  organs  "  (Flexner). 
The  distribution  of  the  virus  as  spray  in  coughing  and  speaking  is  readily 
accomplished  and  by  this  means  both  active  cases  and  passive  carriers  may 
be  produced. 

With  regard  to  other  conceivable  modes  of  propagation  it  is  of  interest  to  note 
that  Rosenau,  whose  observations  have  been  confirmed  by  Anderson  and  Frost, 
has  succeeded  in  conveying  poliomyelitis  from  an  infected  monkey  to  other  monkeys 
by  means  of  the  bites  of  flies  (Stomoxi/s  calcitrans)  but  as  Flexner  points  out  the 
experiment  awaits  convincing  application  to  the  circumstances  surrounding  infection 
in  human  cases  of  the  disease. 

Seeing  that  paralysis  is  not  uncommonly  observed  in  dogs,  hens  and  certain  other 
animals,  it  has  been  thought  possible  that  some  of  the  lower  animals  may  act  as  a 
reservoir  of  the  infection.  So  far  as  experimental  evidence  has  been  obtained  it 
rather  negatives  the  hypothesis  of  a  relationship  between  poliomyelitis  in  man 
and  paralysis  in  the  lower  animals.  These  experiments  "  do  not,  of  course,  exclude 
the  possibility  that  a  reservoir  for  the  virus  may  exist  among  domesticated  animals 
that  do  not  even  respond  to  its  presence  by  developing  paralysis  or  other  conditions 
which  could  be  recognized  as  resembling  poliomyelitis  in  man  "  (Flexner). 

Diagnosis  of  anterior  poliomyelitis. — In  mild  or  atypical  cases  of  the  disease 
which  have  recovered  but  in  which  the  diagnosis  remains  uncertain  informa- 
tion as  to  the  true  nature  of  the  disease  can  be  obtained  by  mixing  some 
serum  from  the  patient  with  the  virus  in  vitro  and  inoculating  an  animal :  a 
control  animal  being  inoculated  with  the  same  dose  of  the  virus  unmixed 
with  serum. 

Acute  anterior  poliomyelitis  and  rabies. — In  many  respects  the  viruses  of  these  two 
diseases  bear  a  close  resemblance  to  one  another.  Thus  they  both  travel  along  the 
nerve  trunks,  both  exhibit  a  selective  affinity  for  the  central  nervous  system,  both 
pass  through  porcelain  or  similar  filters  and  both  react  to  chemical  and  physical 
influences  in  a  similar  manner.  These  resemblances  led  Levaditi  and  Landsteiner 
to  investigate  whether  an  attack  of  one  disease  would  immunize  an  animal  against 
the  other  and  were  able  to  show  that  monkeys  highly  immunized  against  anterior 
poliomyelitis  were  quite  as  susceptible  to  rabies  as  control  animals. 

1  Levaditi  and  Landsteiner,  Ann.  Inst.  Pasteur,  xxiv.  p.  866. 


TYPHUS   FEVER  847 


SECTION  XIII.1— THE  VIRUS  OF  TYPHUS  FEVER.2 

The  micro-organism  which  causes  typhus  fever  in  man  should  be  classified, 
according  to  Ch.  Nicolle,  Conor  and  Conseil,  with  the  filtrable  viruses. 

The  infecting  agent  is  probably  an  intra-cellular  parasite  of  the  white  cells 
of  the  blood.  By  animal  experiment  it  has  been  shown  that  the  blood  is 
infective  from  an  early  stage  of  the  incubation  period  until  convalescence  is 
well  established.  To  obtain  successful  results  with  filtered  blood  the  latter 
should  be  allowed  to  clot  spontaneously  and  the  serum  should  be  filtered 
through  the  most  porous  type  of  Berkefeld  bougie. 

The  results  obtained  by  Nicolle  and  his  co-workers  are  not  in  accordance  with 
the  observations  of  Anderson  and  Goldberger  and  of  Ricketts  and  Wilder  in  America. 
But  as  Nicolle  points  out  the  American  bacteriologists  used  for  inoculation  a  pro- 
duct obtained  by  filtering  the  serum  after  defibrinating  and  then  centrifuging  the 
blood  and  Nicolle  and  his  colleagues,  Conor  and  Conseil,  have  themselves  found 
that  this  product  on  inoculation  into  animals  susceptible  to  infection  with  the  virus 
of  typhus  fever  is  followed  neither  by  infection  of,  nor  by  the  appearance  of  immunity 
in,  these  animals.  And  not  only  so  but  they  find  further  that  the  serum  obtained 
from  blood  which  has  been  allowed  to  clot  spontaneously  though  generally  non- 
infective  after  filtration  is  not  invariably  so.  In  the  opinion  of  Nicolle,  Conor  and 
Conseil  the  only  hypothesis  which  will  explain  their  experimental  observations  is 
that  under  ordinary  conditions  the  amount  of  the  virus  which  passes  the  filter  is 
generally  too  small  to  produce  infection  or  immunity  in  experimental  animals. 

From  the  observations  of  Ricketts  and  Wilder  it  would  appear  that  Mexican 
fever  (El  Tabardillo)  and  typhus  fever  are  due  to  one  and  the  same  micro- 
organism. 

Experimental  infection.  Apes  and  monkeys. — The  chimpanzee  and  some  of 
the  lower  monkeys  (Macacus  sinicus,  M.  rhesus,  M.  cynomolgus,  and  M . 
inuus)  are  susceptible  to  infection  on  inoculation  with  blood  from  human 
cases  of  typhus  fever.  Chimpanzees  are  highly  susceptible  but  the  lower 
monkeys  less  so  ;  moreover  it  would  appear  that  some  individuals  of  a  species 
e.g.  M.  rhesus  are  susceptible  while  others  are  insusceptible. 

In  the  case  of  the  lower  monkeys  in  order  to  ensure  infection  in  susceptible  animals 
not  less  than  4—5  c.c.  of  blood  should  be  inoculated  and  it  is  better  to  inoculate 
intra-peritoneally  than  sub-cutaneously. 

The  incubation  period  varies  both  with  the  species  of  monkey  inoculated  and 
also  with  the  amount  of  blood  used  for  the  experiment.  In  the  chimpanzee  the  incu- 
bation period  is  about  24  days  but  in  the  lower  monkeys  it  is  apparently  shorter 
(13-22  days).  Following  the  incubation  period  the  temperature  rises  (40°  C.  or 
higher)  for  8-10  days  and  then  falls  fairly  rapidly  to  normal.  During  the  febrile 
stage  an  eruption  generally  appears  on  the  face  but  an  injection  of  the  conjunctivas 
may  take  the  place  of  a  rash.  The  animal  is  obviously  unwell  for  the  time  being 
but  about  a  week  after  the  fever  has  disappeared  it  is  restored  apparently  com- 
pletely to  its  normal  health. 

Guinea-pigs. — Guinea-pigs  can  also  be  infected  with  the  virus  of  human 
typhus  fever. 

The  blood  (2-3  c.c.)  should  be  inoculated  into  the  peritoneal  cavity.  The  incuba- 
tion period  varies  from  1—3  weeks  and  the  fever  (40°-41°  C.)  lasts  from  4-9  days. 
The  blood  is  infective  for  monkeys  during  the  whole  of  the  febrile  period. 

In  some  cases  the  inoculated  guinea-pig  may  exhibit  no  rise  of  temperature  but 
the  blood  is  nevertheless  infective. 

Gavino  and  Girard  have  been  able  to  pass  the  virus  through  a  consecutive  series 

1  This  section  has  been  added. 

2Gr.  rC0os  smoke,  mist,  fog.  The  word  was  employed  by  Hippocrates  to  define  a 
confused  state  of  the  intellect  with  a  tendency  to  stupor ;  and  in  this  sense  it  is  aptly 
applied  to  typhus  fever  with  its  slow  celebration  and  drowsy  stupor  (A.  W.  Moore). 


848  THE   FILTRABLE   VIRUSES 

of  eleven  guinea-pigs  and  have  shown  that  the  blood  of  these  animals  is  infective 
for  monkeys. 

Cows,  sheep,  goats,  pigs,  asses,  dogs,  rabbits,  rats  and  fowl  appear  to  be 
naturally  immune  to  the  virus  of  typhus  fever. 

Etiology. — Nicolle,  Comte  and  Conseil  were  able  to  transmit  the  virus  of 
typhus  fever  from  an  infected  Macacus  sinicus  to  two  other  bonnet  monkeys 
by  means  of  body  lice  (Pediculus  vestimenti).  This  observation  has  been  con- 
firmed by  Bicketts  and  Wilder  in  America.  According  to  Goldberger  and 
Anderson  the  virus  can  also  be  transmitted  by  Pediculus  capitis. 

To  ensure  infection  by  means  of  lice  a  considerable  number  of  insects 
should  be  employed.  Ricketts  and  Wilder  have  adduced  evidence  which  goes 
to  show  that  the  virus  of  typhus  fever  multiplies  in  the  bodies  of  lice  and  that 
the  infection  may  possibly  be  transmitted  to  a  second  generation  of  the  insects 
— a  fact  of  considerable  interest  and  importance.  It  would  appear  that  lice 
are  capable  of  infecting  a  new  host  a  week  after  being  fed  upon  an  infected 
individual. 


PART   VII. 

THE  APPLICATION  OF  BACTERIOLOGICAL  METHODS 
TO  THE  EXAMINATION  OF  WATER  AND  AIR. 


3H 


CHAPTER  LXV. 
THE  BACTERIOLOGICAL  EXAMINATION   OF  WATER. 

Introduction. 

Section  I. — The  collection  and  transmission  of  samples  of  water,  p.  851. 

Section  II. — The  methods  of  examination,  p.  853. 

1.  Enumeration  of  the  organism,  p.  853.     2.  Determination  of  the  nature  of  the 
organisms  present,  p.  856.     3.  Houston's  method  of  water  examination,  p.  858. 
The  bacteriological  examination  of  sewage,  p.  861. 

A  CHEMICAL  analysis  of  water  by  showing  the  presence  of  organic  matter, 
nitrites,  chlorides,  ammonia,  etc.,  will  merely  give  a  general  indication  that 
a  water  is  polluted.  A  bacteriological  examination  on  the  other  hand  will  not 
only  reveal  the  fact  that  impurities  are  present  in  a  water  but  will  enable 
the  living  organisms  which  it  may  contain  to  be  enumerated,  isolated  and 
identified.  The  detection  of  pathogenic  micro-organisms  in  water  is  frequently 
of  the  highest  importance. 

The  bacteriological  examination  of  water  may  therefore  be  divided  into 
three  parts  : 

1 .  The  enumeration  of  the  organisms  present. — Quantitative  examination. 

2.  The  determination  of  the  chief  species  present.  1   ^     ,-,    ,- 

3.  The    isolation    and    identification    of   certain}-  ^uantai;lve 

pathogenic  organisms.  [examination. 

With  a  view  to  avoiding  the  introduction  of  organisms  from  without  certain 
precautions  are  necessary  in  collecting  a  water  for  bacteriological  examina- 
tion, and  moreover  the  sample  must  be  transmitted  to  the  laboratory  under 
such  conditions  as  to  prevent  multiplication  of  the  organisms  in  the  water 
before  the  examination  is  begun,  otherwise  the  results  of  the  enumeration 
will  be  falsified. 


SECTION  I.— COLLECTION  AND  TRANSMISSION  OF  SAMPLES 

OF  WATER. 

Collection. — The  water  must  be  collected  in  a  sterile  vessel.  In  the  majority 
of  cases  200-300  c.c.  will  be  sufficient  but  in  certain  cases,  as  for  instance  in 
searching  for  some  of  the  pathogenic  organisms,  it  is  well  to  have  400-500  c.c. 
The  method  of  collection  will  be  as  follows  :— 

1.  Take  a  new  white-glass  bottle  of  suitable  capacity,  rinse  it  out  well 
with  water  and  after  drying  it  plug  it  with  wool  and  sterilize  it. 

2.  To  collect  the  water  for  examination  flame  the  mouth  of  the  bottle, 
remove  the  wool  plug,  fill  the  bottle  as  quickly  as  possible  and  plug  with  a 


852    THE  BACTERIOLOGICAL  EXAMINATION   OF  WATER 


new  tightly-fitting  cork  which  has  been  passed  through  the  flame  of  a  spirit 
lamp  until  slightly  carbonized.  Cut  off  the  cork  level  with  the  mouth  of  the 
bottle  and  seal  with  wax  or — and  this  is  perhaps  better— cover  it  with  a 
sterile  india-rubber  cap.  Paste  a  label  on  the  bottle  giving  the  source  of  the 
sample  and  other  particulars. 

The  method  of  filling  the  bottle  will  depend  upon  whether  the  water  is  to 
be  taken  from  a  tap,  a  river,  a  well,  etc.  Before  taking  a  sample  from  a  tap 
the  water  should  be  allowed  to  run  to  waste  for  several  minutes  in  order  to 
empty  the  pipe  of  any  water  that  may  have  been  standing  in  it.  For  a 
similar  reason,  in  the  case  of  pump  water,  the  water  should  be  pumped  to 
waste  for  10-15  minutes  before  taking  the  sample. 

In  collecting  water  from  a  river  the  bottle  should  be  submerged  with  the 
neck  pointing  up  stream  :  the  sample  should  oot  be  taken  from  too  near  the 
bank  and  the  stirring  up  of  sediment  near  the  place  selected 
should  be  carefully  avoided. 

When  a  well  is  not  provided  with  a  pump  the  bottle  may  be 
lowered  with  string,  or  filled  from  a  bucket  which  must  be 
previously  well  cleansed  and  then  rinsed  with  the  well  water. 
It  is  however  better  in  such  a  case  to  use  a  Miquel's  flask  with 
which  water  can  be  collected  either  from  the  surface  or  from 
any  desired  depth  below  the  surface. 

Miquel's  flask  (fig.  407). — Miguel's  flask  is  a  vessel  of  special  shape  the 
neck  of  which  is  drawn  out  to  a  length  of  5-6  cm.  and  bent  upon  itself. 
Leave  the  pointed  end  unsealed  and  sterilize  the  flask  by  heating  it 
strongly  in  a  flame :  during  the  heating  the  greater  part  of  the  contained 
air  vill  be  driven  out  and  while  still  hot  seal  the  open  end.  When  cold 
the  llask  is  weighted  with  lead  fixed  to  it  by  means  of  iron  wire  and 
can  thus  be  immersed.  A  long  cord  is  attached  to  the  apparatus  to 
allow  of  it  being  lowered  into  a  well ;  and  a  thin  metal  wire  twisted 
round  and  fixed  to  the  narrow  pointed  end  of  the  flask,  long  enough  for 
the  operator  to  have  one  end  always  in  his  hand,  serves  to  break  the 
neck. 

To  take  the  sample  hold  the  cord  and  the  metal  wire  in  one  hand, 
lower  the  flask  into  the  well,  and  when  the  apparatus  has  reached  the 
required  depth  break  the  neck  of  the  flask  by  giving  a  sharp  pull  on  the 
iron  wire.  The  flask  rapidly  fills  with  water  and  as  soon  as  it  is  filled 
raise  it  and  seal  the  open  end  in  a  spirit  flame. 

Other  apparatus. — Miquel's  apparatus  will  usually  be  found  adequate 
but  there  are  many  other  pieces  of  apparatus  designed  for  the   same 
purpose  and  from  among  these  Guillemin's  and  de  Foa's,  which  are 
constructed  in  such  a  way  as  to  ensure  the  automatic  closing  of  the 
flask  as  soon  as  it  is  filled,  may  be  mentioned. 

Transmission. — Organisms  will  rapidly  multiply  in  water  at  the  ordinary 
temperature  of  the  atmosphere  so  that  it  is  necessary  in  the  case  of  water 
intended  for  bacteriological  examination  that  the  sample  should  be  kept  at 
a  temperature  of  about  0°  C.~ a  temperature  which  will  inhibit  multiplica- 
tion— from  the  moment  of  collection  until  the  examination  is  commenced. 

When  the  water  has  to  be  sent  some  distance  the  following  method  of  packing 
may  be  adopted. 

Place  the  bottle  containing  the  water  in  a  metal  box  just  large  enough  to  hold  it 
tightly,  and  for  greater  safety  the  lid  may  be  secured  with  an  india-rubber  band  over 
the  joint.  Place  this  box  in  another  larger  metal  box — a  biscuit  box  does  very 
well — and  fill  the  latter  with  ice  ;  then  place  the  second  metal  box  in  a  larger  wooden 
box,  filling  the  space  between  the  two  with  sawdust  rather  loosely  packed.  This 
arrangement  will  keep  the  sample  of  water  at  the  temperature  of  melting  ice  for 
24-72  hours  depending  upon  the  time  of  year  and  the  amount  of  ice  used.  The 
water  should  invariably  be  sent  to  the  laboratory  by  the  most  rapid  means  of 
transit. 

The  second  metal  box  may  be  dispensed  with  and  in  this  case  the  metal  case  into  which 


FIG.  407.— 
Miquel's  appa- 
ratus for  the 
collection  of 
water  at  differ- 
ent depths. 


COLLECTION   OF  THE   WATER  853 

the  bottle  fits  tightly  is  packed  round  with  ice  and  sawdust — a  large  quantity  of  the 
latter  will  of  course  be  necessary  to  absorb  the  water  from  the  ice  as  it  melts. 

[Houston  has  devised  a  very  convenient  piece  of  apparatus  for  the  collection 
and  transit  of  water  for  bacteriological  examination. 

[It  consists  of  a  rectangular  wooden  box  with  an  hinged  lid  the  whole  lined  with 
thick  felt.  The  box  contains  an  hollow  water-tight  copper  vessel — which  fits  two  sides 
of  the  wooden  box  closely— destined  for  the  reception  of  ice  and  closed  above  with 
a  large  india-rubber  bung.  The  remainder  of  the  space  is  occupied  by  a  felt  box 
open  above  and  divided  into  compartments  for  the  reception  of  flat  rectangular 
bottles.  The  wooden  box  with  its  felt  lining  acts  as  a  non-conductor  of  heat  and  the 
copper  vessel  with  its  contained  ice  keeps  the  temperature  at  as  near  0°  C.  as  possible. 
The  bottles  used  are  ground-glass-stoppered  bottles  of  8  ozs.  capacity.  The  stopper 
should  be  covered  with  paper  before  sterilization  and  this  should  not  be  removed 
until  the  water  is  to  be  collected.  ] 

Particulars  to  accompany  the  sample. — Full  particulars  as  to  the  source 
of  the  water,  the  nature  of  the  examination  required,  etc.  should  accompany 
the  sample  so  that  as  much  information  as  possible  may  be  furnished  upon 
which  to  base  an  opinion. 

The  following  is  a  copy  of  the  official  form  in  use  in  the  French  military  sanitary 
service  : — 

Particulars  of  the  water  sent  for  examination  by — 

(1)  Authority  by  whom  sent — 

(2)  Reasons  for  sending  (epidemic, — spring  to  be  tapped — potable  water, — etc.). 

(3)  Source  of  the  water  (spring,  well,  filtering  gallery,  cistern,  reservoir,  etc.). 
State  the  depth  of  the  well,  cistern,  or  reservoir  and  the  level  of  the  water  at  the 
time  of  collection. 

(4)  Exact  place  where  the  water  was  collected  (e.g.  whether  from  the  spring 
itself  or  from  a  tap  at  the  end  of  a  conduit — whether  from  a  well  or  from  a  pump 
connected  with  the  well) :    never  collect  the  water  which  first  issues  from  a  tap  or 
a  pump.     If  the  water  is  taken  from  a  river,  well  or  reservoir,  state  whether  collected 
from  the  surface,  or  from  the  bottom  or  from  some  intermediate  point.     State  the 
last  occasion  on  which  the  cistern  or  reservoir  was  cleaned  and  whether  there  is  dust 
on  the  surface  or  sediment  at  the  bottom. 

(5)  Has  any  rain  fallen  or  snow  melted  in  the  few  days  preceding  the  taking  of 
the  sample  ?     Is  the  water  muddy  ?     Is  the  level  above  or  below  normal  ? 

(6)  State  any  cause  of  permanent  or  accidental  pollution  to  which  the  water 
appears  to  be  exposed. 

(7)  The  purposes   for  which  the  water  is  required  (i.e.  for  drinking  purposes, 
cooking,  lavatories,  watering  horses,  etc.). 

(8)  Is  the  water  used  for  drinking  without  previous  purification  ?     State,  if  there 
is  any,  the  apparatus  used  for  purification. 

(9)  Atmospheric  temperature  at  the  time  of  collection. 

(10)  The  temperature  of  the  water  at  the  same  time. 

(11)  Day  and  hour  of  collection. 

(12)  Other  remarks. 

SECTION  II.— METHODS   OF  EXAMINATION. 
1.  Enumeration  of  the  organisms. 

Numerous  methods  of  enumerating  the  organisms  in  water  have  been 
suggested.  Some  bacteriologists  base  their  methods  upon  isolation  by 
dilution  in  liquid  media  (p.  76)  (Miquel).  Others  adopt  the  method  of 
isolation  on  gelatin  plates  (Koch). 

It  is  not  proposed  to  discuss  the  pros  and  cons  of  these  different  procedures 
but  those  in  most  general  use  will  now  be  described  in  detail. 

General  rules. — The  cubic  centimetre  is  generally  adopted  as  the  unit  of 
volume  in  enumerating  the  number  of  organisms  in  water  and  it  is  customary 
to  speak  of  a  water  as  containing  say  50,000  organisms  per  c.c. 


854   THE  BACTERIOLOGICAL  EXAMINATION   OF  WATER 

No  account  is  taken  of  the  presence  of  anaerobic  organisms  in  stating  the 
number  present  in  a  water  :  the  determination  is  always  made  under  aerobic 
conditions  and  this  is  always  tacitly  understood.  By  employing  anaerobic 
methods  of  isolation  the  number  of  anaerobic  organisms  could  of  course  be 
determined,  but  in  practice  this  is  never  done,  and  it  presents  difficulties  other 
than  those  of  technique — such  for  instance  as  the  occurrence  of  facultative 
anaerobes,  many  of  which,  as  a  matter  of  fact,  grow  on  the  aerobic  plates. 

Vincent  investigated  the  occurrence  of  anaerobic  organisms  in  water  and  found 
that  the  number  of  strictly  anaerobic  organisms  was  very  small.  He  used  VignaFs 
tubes  and  glucose-gelatin,  containing  sulphindigotate  of  sodium  (p.  92).  The 
medium  was  sown  by  the  dilution  method,  and  for  the  detection  of  pathogenic  species 
the  colonies  so  isolated  were  sub-cultivated  anaerobically  in  broth  and  then  inoculated 
into  animals  (p.  858). 

In  determining  the  number  of  organisms  in  an  unit  volume  of  water  it  is 
usual  to  work  with  a  fraction  of  a  cubic  centimetre  because  the  number  of 
organisms  present  in  a  cubic  centimetre  is  generally  so  large  as  to  make  an 
enumeration  impossible. 

A.  Dilution  method. — 1.  Chip  away  the  wax  with  which  the  cork  was 
sealed  and  flame  the  top  of  the  cork  in  a  Bunsen  :  raise  the  cork  with  a 

flamed  corkscrew  sufficiently  to  allow  of  it 
being  removed  from  the  bottle  with  the 
fingers.1 

2.  Have  ready  on  the  bench  : — 

A  10  c.c.  pipette  graduated  in  1  c.c. 
A  2  c.c.  pipette  graduated  in  0*5  c.c. 
A  drop  pipette  (20  drops  to  1  c.c.). 
(All   of  which   must,    of   course,   have   been 
plugged  with  wool  at  the  upper  end  and  steri- 
lized.) 

A  sterile  glass  vessel  covered  with  paper. 
A  tube  of  sterile  water. 
Several  tubes    of   sterile  gelatin  in  a 
water  bath  at  a  temperature  high 
enough  to  keep  the  medium  fluid. 

'  ?o8r-wa?e?ieJimffion  y°n's)  Several  conical  flasks  plugged  with  wool 

and  sterilized  (fig.  408). 

3.  Into  the  sterile  glass  vessel  measure  9  c.c.  of  sterile  water  adopting  all 
precautions  to  avoid  contamination  :  add  1  c.c.  of  the  water  under  examina- 
tion and  mix  thoroughly.     This  gives  a  dilution  of  1  in  10. 

4.  Flame  the  mouth  of  one  of  the  conical  flasks,  remove  the  wool  plug 
and  with  the  drop  pipette  introduce  2  drops  of  the  1-10  dilution  of  the  water 
(  =  0-01c.c.). 

5.  Flame  the  mouth  of  one  of  the  tubes  of  liquefied  gelatin  (the  tempera- 
ture of  which  should  be  such  that  the  tube  can  be  held  quite  comfortably 
in  the  hand),  remove  the  plug  and  pour  the  contents  quickly  into  the  conical 
flask  (par.  4).     Replace  the  wool  plug  in  the  mouth  of  the  flask,  mix  the 
gelatin  and  water  thoroughly  by  rotating  the  flask  and  then  stand  the  latter 
on  some  cold  and  horizontal  surface  so  that  the  gelatin  may  set  as  quicklv 
as  possible. 

In  effect,  plate  O'Ol  c.c.  of  the  water  on  gelatin. 

[6.  Incubate  at  20°-22°  C.] 

7.  Colonies  will  soon  appear  in  the  gelatin,  each  colony  representing  one 

i  Before  uncorking  the  bottle  shake  it  well  to  mix  thoroughly  the  contents  and  to  dis- 
tribute any  deposit  there  may  be  uniformly  throughout  the  whole. 


QUANTITATIVE   EXAMINATION  855 

organism  originally  present  in  the  water  :  by  counting  the  colonies  the 
number  of  organisms  present  in  the  volume  of  water  sown  can  thus  be  deter- 
mined. The  flask  must  be  examined  daily ;  the  best  way  is  to  invert  it ; 
the  colonies  are  then  visible  in  the  transparent  medium  through  the  bottom 
of  the  flask,  and  can  be  readily  counted. 

Thus,  if  on  the  third  day,  twelve  colonies  are  visible  a  note  should  be 
made. 

3rd  day.  5  August.  12  colonies. 

To  avoid  counting  the  same  colonies  twice,  mark  their  position  with  a  pen 
and  ink  on  the  base  of  the  flask  as  they  are  counted. 

If  next  day  in  addition  to  the  twelve  colonies  marked  eight  more  have 
appeared  the  note  will  read 

3rd  day.  5  August.  12  colonies 

4th  day.  6  August.  20  colonies 

and  so  on. 

The  enumeration  is  ordinarily  completed  by  the  fifteenth  to  the  twentieth 
day,  after  which  no  new  colonies  are  likely  to  appear.  Suppose  then  that  the 
result  is 

20th  day.  22  August.  64  colonies 

the  number  of  aerobic  organisms  in  a  cubic  centimetre  of  the  water  will  be 
ascertained  by  multiplying  64  by  100.  The  water  therefore  is  said  to  contain 
6400  organisms  per  c.c. 

It  is  as  well  to  sow  several  flasks  with  every  sample  of  water,  which  may  be  num- 
bered 1,  2,  3  and  so  on ;  the  mean  of  the  various  results  will  be  nearer  the  truth 
than  the  result  of  a  single  determination  is  likely  to  be :  and  if  as  often  happens 
the  number  of  organisms  in  the  flasks  varies  but  little,  the  accuracy  of  the  technique 
is  demonstrated  and  the  results  are  of  greater  value. 

In  the  case  illustrated  it  is  assumed  that  no  liquefaction  of  the  gelatin  has 
occurred  to  interfere  with  the  enumeration.  Unfortunately,  however,  this 
is  not  the  usual  experience  in  practice  ,  it  is  much  more  common  to  find  that 
the  water  contains  a  high  percentage  of  "  liquefying  "  organisms  so  that 
after  the  first  few  days  further  enumeration  is  rendered  difficult  and  finally 
impossible.  In  such  cases  the  count  should  be  continued  until  the  plate  is 
entirely  liquefied  and  then  the  date  of  liquefaction  noted.  Thus,  for  instance, 
2nd  day,  -  26  colonies 

3rd      „  59         „ 

4th      „       -  102 

5th      „       -  Plate  entirely  liquefied 

the  results  of  the  analysis  should  be  recorded  thus 

10,200  (102  x  100)  aerobic  organisms  per  cubic  centimetre.  This  number  is 
much  beloiv  the  real  total,  liquefaction  of  the  gelatin  having  terminated  the  count 
on  the  fifth  day. 

The  statement  of  the  result  must  be  qualified  in  this  way  whenever  liquefac- 
tion occurs  before  about  the  tenth  day. 

Note. — Many  waters  contain  moulds  as  well  as  bacteria.  The  moulds 
develop  on  the  plates  and  must  be  separately  enumerated.  Thus,  for  instance, 
a  sample  of  water  might  contain 

1256  aerobic  bacteria  and  300  moulds  per  cubic  centimetre. 

B.  Method  of  enumeration  using  a  -V  c-c-  pipette.— The  dilution  method 
is  not  only  rather  tedious  but  in  inexpert  hands  affords  opportunities  of  con- 
tamination. The  method  now  about  to  be  described  is  therefore  often 
preferred. 

The  pipettes  made  by  Alvergniat  should  be  used  :  they  are  very  carefully 
calibrated  and  give  about  50  drops  to  the  cubic  centimetre.  Pipettes  giving 


856   THE  BACTERIOLOGICAL  EXAMINATION  OF  WATER 

exactly  50  drops  to  the  cubic  centimetre  cannot  always  be  obtained  on 
account  of  the  difficulties  in  their  manufacture  but  the  actual  number — 48, 
52,  54  drops  or  whatever  the  number  may  be — is  always  marked  on  the  stem. 

Sterilize  a  pipette  (which  it  will  be  assumed  gives  52  drops  to  the  cubic 
centimetre).  Aspirate  a  little  of  the  water  under  examination  and  introduce 
one  drop  into  a  conical  flask  :  add  the  gelatin  and  proceed  with  the  enumera- 
tion as  above  (from  par.  5). 

Then  by  multiplying  the  number  of  colonies  which  have  developed  in  the 
flask  by  52  the  number  of  organisms  per  cubic  centimetre  can  be  ascertained. 
If,  for  instance  96  colonies  were  counted 

96  x  52  =  4992  aerobic  organisms  per  c.c. 

Two  or  three  flasks  should  always  be  sown  and  the  average  of  the  results 
recorded. 

Interpretation  of  the  results  of  enumeration. 

The  quantitative  examination  is  not  alone  sufficient  to  allow  an  opinion 
to  be  given  on  the  purity  of  a  water  ;  before  drawing  any  conclusion  a  qualita- 
tive examination — a  determination  of  the  nature  of  the  organisms  present — 
must  be  undertaken.  It  is  at  once  apparent,  for  instance,  that  a  water  con- 
taining a  large  number  of  harmless  saprophytes  (B.  subtilis,  the  white  coccus 
of  water,  etc.)  would  be  infinitely  preferable  to  another  which  contained  a 
few  pathogenic  organisms  such  as  the  typhoid  bacillus.  Still,  from  the  point 
of  view  of  ordinary  pollution  the  total  number  of  organisms  is  of  some  import- 
ance. Miquel  has  classified  waters  according  to  their  content  of  organisms  ; 
this  classification  is  convenient  but  it  should  not  be  utilized  until  the  results 
of  the  qualitative  examination  are  known  and  have  been  taken  into  con- 
sideration. 

MIQUEL'S  TABLE. 


0  to  10        organisms  per  cubic  centimetre, 

10    „  100 

100    „  1,000 

1,000    „  10,000 

10.000    „  100,000 

More  than  100,000 


Extraordinarily  pure. 

Very  pure. 

Pure. 

Moderate. 

Impure. 

Very  impure. 


Note. — Any  such  classification  as  this  is,  of  course,  very  arbitrary  and  of  limited 
value  for  the  reasons  given.  It  may  be  remarked  here  that  most  methods  of  enumera- 
tion are  liable  to  underestimate  the  numbers  of  organisms  contained  in  a  given 
volume  of  water.  For  instance,  there  may  be  organisms  present  in  the  water  which 
fail  to  grow  on  the  culture  medium  on  account  of  the  presence  of  other  species  acting 
on  them  prejudicially  as  "  inhibiting  organisms."  Moreover,  pathogenic  species  of 
organisms  will  hardly  grow  on  gelatin  plates,  not  only  because  they  are  held  in  check 
by  saprophytes,  but  also  because  the  temperature  of  incubation  is  unsuited  to  their 
multiplication.  It  will  be  obvious  that  Miquel' s  dilution  method  in  which  an 
attempt  is  made  to  sow  each  organism  in  a  different  tube  is  free  from  these  sources 
of  error,  but  unfortunately  by  reason  of  its  complexity  it  is  of  little  practical  value. 

QUALITATIVE   EXAMINATION. 

2.  Determination  of  the  nature  of  the  organisms  present. 
A.  The  isolation  of  saprophytic  species. 

It  is  usual  for  the  identification  of  the  various  species  of  micro-organisms 
which  may  be  present  in  a  water  to  isolate  the  organisms  on  Petri  dishes 
(p.  78).  Sow  one  drop  of  the  water  in  a  tube  of  melted  gelatin,  mix.  sow 
two  or  three  loopsful  of  the  mixture  into  a  second  tube  of  gelatin,  mix 
again,  and  sow  two  or  three  loopsful  from  the  second  tube  into  a  third  tube 
and  pour  plates  with  the  mixtures.  The  growths  on  the  plates  are  carefully 


QUALITATIVE   EXAMINATION  857 

watched  and  sub-cultures  sown  when  necessary  for  the  identification  of  a 
particular  colony. 

In  the  case  of  beginners  the  identification  of  the  various  micro -organic 
species  involves  a  very  considerable  amount  of  labour  :  each  colony  must 
be  separately  examined  with  the  naked  eye  and  under  the  microscope,  the 
morphology  of  the  organism  must  be  studied  and  its  cultural  characteristics 
investigated  together  with  its  effect  on  animals.  A  little  practice  however 
soon  confers  sufficient  knowledge  to  enable  the  greater  number  of  the  colonies 
to  be  recognized  quite  easily. 

It  is  not  within  the  province  of  this  book  to  enter  upon  a  detailed  description  of 
the  saprophytic  micro-organisms  likely  to  be  encountered  in  water,  but  it  may  be 
said  that  while  some  of  them  are  absolutely  harmless  others  (e.g.  Proteus  vulgaris, 
Micrococcus  prodigiosus)  elaborate  soluble  products  which  may  give  rise  to  symptoms 
of  toxaemia  in  man  and  the  lower  animals.  These  species  tend  to  inhabit  decom- 
posing animal  matter  so  that  their  isolation  from  a  water  to  be  used  for  domestic 
purposes  would  be  an  unfavourable  indication.  The  odour  again  which  is  given 
off  from  the  gelatin  plates  should  be  noted  for  it  is  not  unusual  to  find  that  bacteria 
associated  with  putrefactive  processes  give  rise  to  disagreeable  ammoniacal 
emanations. 

B.  The  detection  of  pathogenic  species. 

As  has  been  said  above  the  gelatin-plate  method  is  not,  speaking  generally, 
an  efficient  method  for  the  detection  of  pathogenic  species,  and  for  the  isola- 
tion of  such  organisms  the  author  has  adopted  for  several  years  now  with 
considerable  success  the  following  technique  which  he  recommends  should 
form  an  integral  part  of  every  water  examination. 

Sow  0*5-2  c.c.  of  the  water  in  tubes  of  broth  or  better  Metchnikoff's  liquid 
peptone-gelatin  medium  and  incubate  the  tubes  forthwith  at  38°  C.  In 
some  cases  no  growth  whatever  will  be  visible  after  24  hours  :  the  experi- 
ment need  not  then  be  pursued  further,  and  the  results  may  be  regarded  as 
negative.  Much  more  frequently  however  a  cloudiness  will  appear  in  the 
tubes  after  incubating  for  5-8  hours.  This  rapidly-appearing  turbidity  is 
almost  always  due  either  to  the  colon  bacillus  or  to  other  species  of  pathogenic 
organisms,  since  saprophytic  bacteria  grow  more  slowly  at  this  temperature. 
When  the  cloudiness  is  well-marked  within  6-10  hours  of  sowing  transfer  a 
loopful  of  fluid  to  a  fresh  tube  of  medium  and  after  mixing  sow  a  loopful  into 
a  second  tube  and  incubate  again.  As  soon  as  the  second  tube  becomes 
cloudy  plate  a  trace  of  the  growth  on  agar  by  the  parallel  stroke  method  for 
the  purpose  of  getting  single  colonies.  Incubate  the  plates  in  a  moist 
chamber  at  37°  C.  which  affords  conditions  particularly  favourable  to  the 
rapid  multiplication  of  pathogenic  organisms.  This  method  has  enabled  the 
author  to  isolate  from  different  waters  Bacillus  pyocyaneus,  the  pneumo- 
bacillus  of  Friedlander,  the  colon  bacillus  and  the  various  micro-organisms 
of  suppuration. 

The  isolation  of  the  colon  bacillus  or  of  closely  related  bacteria  from  a 
water  is  a  matter  of  frequent  occurrence  and  formerly  so  much  importance 
was  attached  to  the  fact  that  any  water  in  which  it  was  found  was  con- 
demned. At  the  present  time  however  since  with  more  perfected  methods 
the  colon  bacillus  can  be  isolated  from  a  very  large  number  of  waters  there 
is  a  tendency  to  go  to  the  opposite  extreme,  to  attach  no  importance  what- 
ever to  its  occurrence  and  to  regard  it  as  an  harmless  saprophyte.  In  the 
author's  opinion  the  truth  lies  between  these  extremes,  for  though  in  some 
cases  the  presence  of  a  few  colon-like  bacteria  may  be  without  significance, 
in  other  cases  it  may  indicate  direct  pollution  with  matter  of  excretal  origin. 
Further  it  must  not  be  forgotten  that  this  organism  is  found  in  a  large 


858   THE   BACTERIOLOGICAL   EXAMINATION   OF   WATER 

number  of  samples  of  typhoid -infected  waters  and  it  may  altogether  mask 
the  presence  of  the  typhoid  bacillus. 

Whenever  a  colon  bacillus  is  isolated  from  a  sample  of  water  its  charac- 
teristics should  be  carefully  studied.  If  these  agree  in  all  details  with  those 
of  the  typical  colon  bacillus  of  Escherich  (and  the  author  attaches  considerable 
importance  to  the  rapidity  with  which  milk  is  coagulated)  and  more  particu- 
larly if  the  strain  isolated  prove  to  be  pathogenic  to  guinea-pigs  (using  O5-1 
c.c.  of  a  24-hour  broth  culture  intra-peritoneally)  no  hesitation  need  be  felt 
in  expressing  an  adverse  opinion  upon  the  water.  The  coincident  occurrence 
of  bacteria  associated  with  putrefaction  renders  it  still  more  probable  that 
the  pollution  is  excretal  in  origin. 

Great  importance  is  to  be  attached  to  the  inoculation  of  animals  with 
organisms  isolated  from  water  and  grown  at  37°  C.  This  experiment  should 
never  be  omitted  before  coming  to  a  final  conclusion. 

Vincent  attaches  some  importance  to  the  numbers  in  which  the  colon  bacillus  is 
found  in  a  water  and  in  his  opinion  a  water  which  only  contains  10  to  50  colon  bacilli 
per  litre  may  be  regarded  as  good  in  quality.  For  the  purpose  of  ascertaining  the 
number  present  he  adopts  the  dilution  method  using  broth  containing  0'70  per  1000 
of  carbolic  acid. 

[An  opinion  upon  a  water  must  be  based  upon  a  number  of  data  of  which 
the  occurrence  of  the  colon  bacillus  is  merely  one.  The  numbers  in  which 
the  colon  bacillus  or  colon-like  bacteria  are  present  is,  of  course,  a  matter  of 
importance  but  its  importance  depends  upon  other  factors  :  for  these  numbers 
will  vary  according  to  the  source  of  the  water,  and  a  number  which  would  be 
sufficient  to  condemn  absolutely  a  deep  well  water  would  be  disregarded  in 
an  upland  surface  water.  In  effect  an  absolute  standard  cannot  be  fixed  : 
a  mere  laboratory  examination  of  the  water  without  a  full  knowledge  of 
its  source  is  in  the  vast  majority  of  cases  of  little  or  of  no  value.  The  real 
value  of  a  bacteriological  examination  will  depend  upon  the  consideration  of 
all  the  facts  both  as  to  source,  method  of  collection,  mode  of  transit,  and  the 
nature  and  number  of  the  various  micro-organic  species  present ;  and  the 
interpretation  of  these  data  is  a  matter  requiring  considerable  knowledge 
and  experience.  Moreover  it  is  now  agreed  by  all  who  are  in  a  position  to 
express  an  opinion,  that  before  a  reliable  conclusion  as  to  the  purity  of  a 
water  can  be  arrived  at  the  laboratory  examination  must  be  systematic 
extending  over  a  period  sufficient  to  cover  all  possible  or  likely  sources  of 
contamination.] 

C.  Systematic  examination  for  certain  pathogenic  organisms. 

During  an  epidemic  of  enteric  fever  or  cholera  or  if  a  number  of  cases  of 
anthrax  occur,  the  specific  organisms  should  be  systematically  sought  for. 

The  pathogenic  organisms  which  have  most  frequently  to  be  isolated  are 
the  colon  bacillus,  the  typhoid  bacillus,  the  pneumobacillus,  the  anthrax 
bacillus  and  the  cholera  vibrio.  The  methods  to  be  adopted  in  each  of  these 
cases  have  already  been  described  in  the  chapters  devoted  to  a  detailed 
consideration  of  these  organisms. 

3.  Houston's  method  of  water  examination.1 

Houston   some  years  ago  introduced   a   method  for  the   bacteriological 
examination  of  water  which  in  the  United  Kingdom  and  in  many  parts  of 
the  Dominions  beyond  the  Seas  has  now  superseded  all  others. 
The  sample  of  water  is  examined  with  a  view  to  determining 
1.  The  total  number  of  organisms  present. 

1  This  sub-section  has  been  added. 


HOUSTON'S   METHOD   OF   WATER   EXAMINATION       859 

2.  The  presence  of  the  colon  bacillus  and  of  colon-like  bacteria  and  the 

approximate  numbers  of  these  organisms. 

3.  The  presence  of  the  Bacillus  enteritidis  sporogenes  and  the  numbers 

in  which  it  occurs. 

4.  The  presence  of  streptococci. 

1.  The  total  number  of  organisms  present  is  ascertained  by  methods  similar 
to  those  described  above  (Section  II.  1.)  and  both  agar  and  gelatin  plates 
may  be  sown  to  ascertain  the  numbers  of  organisms  which  will  grow  at  37°  C. 
and  22°  C.  respectively. 

2.  The  detection  of  the  colon  bacillus. — For  this  determination  Houston 
uses  MacConkey's  bile-salt  fluid   (p.   412)   as  the  preHminan^  enrichment 
medium  and  then  MacConkey's  agar  medium  for  isolation  from  the  pre- 
liminary medium. 

3.  The  presence  of  the   Bacillus  enteritidis  sporogenes  is  determined  by 
sowing  known  volumes  of  the  water  in  milk  and  cultivating  anaerobically. 

4.  For  the  detection  of  streptococci  measured  quantities  of  the  water  are 
sown  in  broth  and  incubated  at  37°  C. 


Experimental  data. 

1.  Apparatus  and  media  required. 

(a)  Pipettes  graduated  to  measure  (1)  100  c.c.  (2)  10  c;c.  (3)  1  c.c.  (4)  O'l  c.c. 
(/3)  Media. 

(1)  Test  tubes  containing  a  measured  9  c.c.  of  sterile  distilled  water. 

(2)  Tubes  containing  about  10  c.c.  of  sterile  gelatin  and  others  containing 

about  10  c.c.  of  sterile  agar. 

(3)  Tubes   containing   about   10   c.c.    of  MacConkey's  lactose-bile-salt 

fluid1  (p.  412). 

(4)  Tubes  containing  a  measured  10  c.c.  of  MacConkey's  fluid  of  double 

strength  : — 

Peptone,     -  40  grams. 

Lactose.      -  10      „ 

Sodium  taurocholate,  -  10       „ 

Chalk,1        ....  20      „ 

10  per  cent,  litmus  solution,  -  -          -         200  c.c. 

Water,         ...  ...       1000    „ 

(5)  Tubes  containing  a  measured  50  c.c.  of  MacConkey's  fluid  of  triple 

strength  : — 

Peptone,     -  60  grams. 

Lactose,      -  20      „ 

Sodium  taurocholate,  -                                                                     15       „ 

Chalk,         -  30      „ 

10  per  cent,  litmus  solution,  300  c.c. 

Water,         -         -  1000    „ 

(6)  Tubes  containing  about  (i)  50  c.c.  and  (ii)  10  c.c.  of  sterilized  milk 

from  which  the  cream  has  not  been  removed. 

(7)  Tubes  containing  about  10  c.c.  of  sterile  broth. 

(8)  Petri  dishes. 

2.  Technique. 

Remove  the  bottle  containing  the  sample  of  water  from  its  case  and 
thoroughly  mix  the  contents  by  shaking.  Take  out  the  stopper  and  flame 
the  mouth  of  the  bottle. 

1.  Sow  100  c.c.  into  50  c.c.  of  MacConkey's  triple  strength  fluid. 

1  Chalk  is  added  in  order  that  the  acid  and  gas  produced  by  lactose-fermenting  organisms 
may  be  more  readily  recognized.  Some  observers  prefer  a  small  thin-walled  glass  tube 
(Durham's  tube)  which  is  inverted  into  the  medium  and  serves  to  collect  the  gas. 


860   THE   BACTERIOLOGICAL  EXAMINATION   OF  WATER 

2.  Sow  10  c.c.  into  10  c.c.  of  MacConkey's  double  strength  fluid  and  a 
second  10  c.c.  into  50  c.c.  of  milk  being  careful  that  the  water  passes  beneath 
the  cream. 

3.  Sow  1  c.c.  into  a  tube  of  MacConkey's  medium  (ordinary  strength) ;   a 
second  1  c.c.  into  10  c.c.  of  milk  ;  a  third  1  c.c.  into  broth  and  a  fourth  into 
9  c.c.  of  sterile  water. 

4.  Thoroughly  mix  the  last  tube  in  which  the  water  under  examination 
has  been  added  to  sterile  water.     Sow  1  c.c.  of  this  mixture — representing 
0*1  c.c.  of  the  water — into  MacConkey's  fluid,  into  gelatin  and  into  agar 
(melted  and  cooled  to  40°  C.)  and  a  further  1  c.c.  into  a  second  tube  of  sterile 
water  (9  c.c.). 

5.  After  mixing  use  1  c.c.  of  the  diluted  water — equivalent  to  O'Ol  c.c.  of 
the  water — for  sowing  MacConkey's  medium,  gelatin  and  agar  as  before  ; 
and  if  necessary  proceed  to  further  dilution. 

6.  Pour  the  agar  and  gelatin  into  Petri  dishes.     Incubate  the  cultures  at 
37°  C.  with  the  exception  of  the  gelatin  plates  which  must  be  grown  at  22°  C. 
The  milk  tubes  must  be  heated  first  at  80°  C.  for  15  minutes  and  then  be 
grown  anaerobically  in  a  Bulloch's  apparatus  (p.  96). 

The  plates  must  be  examined  daily  and  the  colonies  counted  as  described 
above.  The  other  cultures  are  examined  after  48  hours'  incubation : — 

i.  The  broth  tubes  for  streptococci  by  examining  the  deposit  micro- 
scopically. 

ii.  The  milk  tubes  for  the  "  enteritidis  change" — considerable  formation 
of  gas,  an  odour  of  butyric  acid,  separation  of  the  curd  from  the  whey  and 
tearing  up  of  the  curd  by  the  gas  evolved. 

iii.  The  MacConkey's  tubes  for  the  presence  of  gas. 

If  gas  is  formed  a  loopful  of  the  culture  is  diluted  in  sterile  water  and  one 
or  two  loopsful  of  the  dilution  used  for  sowing  a  surface  culture  of  MacConkey's 
taurocholate-lactose-agar  for  the  purpose  of  isolation.  The  agar  is  incubated 
at  37°  C.  and  after  24  or  48  hours  is  examined  for  colon-like  colonies.  If 
such  colonies  be  found  one  or  more  is  sown  in  a  tube  or  tubes  of  liquefied 
glucose-gelatin  and  the  tubes  incubated  at  20°  C.  If  gas  be  formed  the 
gelatin  is  liquefied  and  the  culture  used  for  sowing  the  following  media  : — 

1.  Neutral-red  broth. 

1  per  cent,  solution  of  neutral-red,  2  c.c. 

Broth,         ...  .  1000     „ 

2.  Peptone  water. 

3.  Litmus  milk. 

These  tubes  are  incubated  at  37°  C.  and  then  examined  respectively  for 

i.  Fluorescence  in  the  neutral-red-broth. 

ii.  Indol  in  peptone  water  (Ehrlich's  test,  p.  374  e). 

iii.  Acid  and  clot  in  milk. 

If  an  organism  which  gives  all  the  reactions  described  has  been  recovered 
from  any  of  the  MacConkey  tubes  the  water  is  said  to  contain  "  typical  colon 
bacilli " l  in  that  amount.  Thus  if  an  organism  having  these  characteristics 
be  isolated  from  the  100  c.c.  tube  but  not  from  the  10  c.c.,  1  c.c.  or  O'l  c.c.  tubes 
the  water  is  said  to  contain  typical  colon  bacilli  in  100  c.c,  but  not  in  less. 
Similarly  a  water  from  1  c.c.  of  which  a  typical  colon  bacillus  was  isolated 
but  not  from  O'l  c.c.  is  said  to  contain  at  least  1  but  not  10  colon  bacilli 
per  c.c. 

1Such  an  organism  is  described  by  Houston  as  a  "  Flaginac  "  colon  bacillus: — Fl, 
fluorescence  in  neutral-red-broth  ;  ag,  acid  and  gas  in  a  lactose  medium  ;  in,  indol  in 
peptone  water  ;  ac,  acid  and  clot  in  milk. 


THE   BACTERIOLOGICAL  EXAMINATION   OF  SEWAGE     861 


To  give  some  concrete  notions  with  regard  to  the  nature  and  number  of 
organisms  found  in  water  some  figures  may  be  abstracted  from  Dr.  Houston's 
reports.  Taking  the  London  Metropolitan  water  supply  for  the  year  ending 
March  31st  1909  :— 

1.  Total  number  of  organisms  (Gelatin  plates  at  20°-22°  C.  3  days). — 


River  Thames. 

River  Lea. 

New  River. 

Raw  water  (organisms  per  c.c.)    - 

2558 

8794 

1118 

Filtered  water  (organisms  per  c.c.) 

11*3 

18-9 

6-1 

2.  "  Typical  "  colon  bacillus  test. — 


River  Thames. 

River  Lea. 

Xew  River. 

All  London 
Waters. 

Raw  waters  - 

15  per  c.c. 

25  per  c.c. 

1  per  c.c. 

— 

Filtered  waters  - 

— 

— 

— 

2  per  litre. 

The  bacteriological  examination  of  sewage. 

[Houston's  method  of  water  examination  is  equally  applicable  to  the 
investigation  of  the  nature  and  number  of  organisms  present  in  sewage.  In 
this  case,  of  course,  the  sewage  must  be  diluted  to  a  far  greater  extent  than 
is  necessary  in  the  case  of  water  and  it  will  be  necessary  to  examine  0000,001, 
0-000,000,1  and  even  0-000,000,01  c.c.  for  the  presence  of  colon  bacilli.] 


CHAPTER  LXVI. 
THE  BACTERIOLOGICAL  EXAMINATION  OF  AIR. 

Introduction. 

1.  Original  methods,  p.  862. 

2.  Methods  employed  at  the  present  day,  p.  864. 

A.  Methods  based  upon  nitration,  p.  865. 

B.  Methods  based  upon  bubbling  the  air  through  a  suitable  liquid,  p.  866. 

THE  bacteriological  examination  of  air  may  be  either  quantitative  or 
qualitative,  depending  upon  whether  it  is  proposed  to  ascertain  the  number 
of  organisms  present  in  a  given  volume  of  air  or  whether  the  object  is  to 
determine  to  what  species  these  organisms  belong.  Finally,  the  object  of  the 
experiment  may  be  to  detect  the  presence  of  some  given  pathogenic  organism. 
Since  the  number  of  organisms  in  air  is  small  the  unit  adopted  is  a  cubic 
metre  :  thus  it  is  usual  to  say  that  the  air  of  a  room  contains  500,  1000,  or 
3000  organisms  per  cubic  metre  as  the  case  may  be. 

For  a  long  time  it  was  considered  sufficient  to  examine  microscopically  the  dust 
of  the  air  collected  by  means  of  an  aeroscope.  The  aeroscope  most  generally  in  use 
in  France  is  that  of  Pouchet.  It  consists  of  a  glass  cylinder  closed  at  both  ends  : 
inside,  about  the  middle,  an  ordinary  microscope  slide  is  held  by  two  supports  and 
on  the  upper  surface  of  the  slide  a  drop  of  glycerin  is  placed.  The  top  of  the  glass 
cylinder  is  perforated  in  the  centre  by  a  circular  hole  carrying  a  platinum  funnel, 
the  tube  of  which  passes  into  the  cylinder  above  the  centre  of  the  slide.  A  tubulure 
fixed  to  the  lower  part  of  the  aeroscope  is  connected  to  an  aspirator.  When  the 
aspirator  is  working,  the  air  passing  through  the  funnel  impinges  upon  the  slide 
and  deposits  its  suspended  dust  which  is  retained  there  by  the  glycerin.  When 
a  sufficient  volume  of  air  has  been  drawn  through,  the  aspirator  is  turned  off, 
and  the  slide  removed  ;  the  dust  is  distributed  by  means  of  a  sterile  needle  through 
the  glycerin  which  is  then  covered  with  a  cover-glass  and  examined  under  the  micro- 
scope. In  this  way  the  larger  particles  of  dust  in  the  air  may  be  studied — spores 
of  fungi,  moulds,  pollen,  starch  grains,  mineral  particles,  etc. — but  the  method  is 
not  sufficiently  delicate  for  the  detection  of  bacteria  and  their  spores.  At  the  present 
time  cultural  methods  are  employed  practically  to  the  exclusion  of  all  others. 

1.  Original  methods. 

I.  Pasteur's  method. — Pasteur's  method  which  is  the  oldest  of  all  consists  in  the 
use  of  long-necked  flasks  one-third  filled  with  veal  broth.  The  neck  of  each  flask 
is  drawn  out  to  a  point,  the  flask  and  its  contents  are  sterilized  and  the  point  sealed 
in  a  small  flame  while  the  broth  is  still  at  the  boiling  point :  in  this  way  all  the  air 
is  driven  out.  It  is  now  only  necessary  to  carry  the  flask  to  the  place  where  the  air 
is  to  be  examined  and  to  break  off  the  fine  point.  The  air  with  the  particles  in 
suspension  will  rush  into  the  flask,  and  as  soon  as  it  is  full  the  point  is  sealed  again, 


ORIGINAL  METHODS 


863 


and  the  flask  put  on  one  side  :  the  same  process  is  repeated  with  a  number  of  flasks. 
In  a  short  time  the  medium  in  some  of  the  flasks  will  become  cloudy,  and  from  the 
number  which  show  this  turbidity  the  number  of  organisms  contained  in  the  air 
can  be  roughly  deduced. 

Thus,  for  example,  suppose  that  50  flasks  each  containing  approximately  500  c.c.  of 
air  were  used  and  that  20  of  them  became  cloudy  :  the  calculation  would  be  as  follows  : — 
25  litres  of  air  have  given  20  organisms ;  1  cubic  metre  therefore  contains  very  approxi- 
mately f§  x  1000  that  is  800  organisms. 

The  method  necessitates  the  use  of  a  great  deal  of  material  and  is  cumbersome 
and  from  the  practical  point  of  view,  impossible. 

II.  Konh's  method. — Koch's  method  consists  in  exposing  to  the  air  for  different 
periods  of  time  a  number  of  gelatin  plates  and  studying  the  colonies  which  subse- 
quently develop  on  them.  This  method  does  not  allow  of  quantitative  estimations. 


FIG.  409. 


FIG.  410. 


FIGS.  409,  410. — An  agar  plate  and  a  gelatin  plate  exposed  to  the  air  side  by 
side  and  then  incubated  at  37°  C.  and  20°  C.  respectively.  Note  the  greater 
number  of  bacteria  on  the  former  and  of  moulds  on  the  latter. 

III.  Hesse's  method. — Hesse's  method  which  has  the  advantage  of  being  simple 
is  based  upon  the  principle  of  the  aeroscope.  Unfortunately  the  results  obtained 
are  only  approximate. 

Take  a  piece  of  glass  tubing  4-5  cm.  in  diameter  and  50-70  cm.  long  (fig.  411). 
Plug  one  end  of  the  tube  with  an  india-rubber  plug  through  which  a  piece  of  glass 
tubing  plugged  with  wool  at  the  outer  end  is  passed. 


FIG.  411.— Hesse's  tube. 


Cover  the  other  end  with  two  pieces  of  india-rubber  one  over  the  other  the  inner 
being  perforated  with  a  hole  about  1  cm.  in  diameter.  Sterilize  the  apparatus  and 
then  pour  about  50  c.c.  of  liquefied  sterile  gelatin  into  the  tube  through  the  perforated 
piece  of  india-rubber.  Replace  the  second  sheet  of  india-rubber  at  once  and  keep 
the  tube  in  an  horizontal  position  until  the  gelatin  has  set.  The  gelatin  should  form 


864       THE  BACTERIOLOGICAL  EXAMINATION   OF  AIR 

a  smooth  uniform  layer  on  the  lower  surface  of  the  tube  not  deep  enough  to  reach 
the  orifice  of  the  small  glass  tube  nor  the  opening  in  the  india-rubber  at  the  other 
end.  [It  will  be  found  more  satisfactory  to  warm  the  tube  before  pouring  the 
gelatin  into  it  and  then  holding  it  horizontally  to  rotate  the  tube  until  the  gelatin 
has  set  so  that  the  medium  forms  a  thin  coating  over  the  whole  of  the  interior.] 
The  apparatus  is  now  ready  for  use.  When  about  to  carry  out  an  experiment, 
remove  the  outer  piece  of  india-rubber,  attach  the  outer  end  of  the  small  glass  tube 
to  an  aspirator  and  draw  10-15  litres  of  air  slowly  through  the  tube.  The  air  enters 
the  hole  in  the  india-rubber  capsule  and  passes  over  the  surface  of  the  gelatin  on  which 
it  is  intended  that  the  suspended  dust  should  be  deposited.  When  the  required 
volume  of  air  has  been  drawn  through,  the  outer  india-rubber  capsule  is  replaced 
and  the  tube  incubated  in  the  cool  incubator  (20°  C.).  Colonies  begin  to  appear 
on  the  gelatin  in  the  course  of  a  day  or  two  and  if  the  technique  was  satisfactory 
should  be  more  numerous  at  the  end  at  which  the  air  entered.  The  colonies  can 
be  counted  and  any  which  it  may  be  desired  to  investigate  further,  picked  off. 

If,  for  example,  15  litres  of  air  have  been  aspirated  and  6  colonies  of  bacteria  and  10 
moulds  are  subsequently  counted  on  the  gelatin  the  air  will  have  contained  approximately 
T\  x  1000  =  400  aerobic  bacteria  per  cubic  metre. 
|2  x  1000  =  666  moulds  per  cubic  metre. 

In  practice,  however,  it  happens  that  many  organisms  stick  to  the  glass  wall  of 
thtf  tube  and  so  do  not  enter  into  the  computation  [this  source  of  error  is  avoided  if 
the  medium  be  coated  over  the  whole  surface] :  further,  if  the  experiment  be  con- 
tinued for  any  length  of  time  the  gelatin  will  become  dry  and  fail  to  act  as  a  satis- 
factory culture  medium  •  and  lastly,  the  current  of  air  must  pass  very  slowly  other- 
wise the  organisms  suspended  in  it  will  be  carried  through  the  tube  without  being 
deposited.  [Two  other  objections  may  be  raised,  namely  the  difficulty  of  reaching 
the  colonies  should  it  be  desirable  to  sub-cultivate  them  and  the  fact  that  some 
organisms  rapidly  liquefy  the  gelatin  and  render  the  experiment  useless.] 

2.  Methods  employed  at  the  present  day. 

The  methods  just  described  have  now  been  superseded  by  others  which 
depend  upon  removing  the  organisms  contained  in  the  air  either  by  bubbling 
the  latter  through  a  viscous  fluid  or  by  filtering  it  through  a  powder.  By 
adopting  either  of  these  methods  all  the  organisms  suspended  in  a  given 
volume  of  air  can  be  collected  in  a  small  space,  being  either  disseminated 
in  the  liquid  or  mixed  with  the  powder  as  the  case  may  be.  It  will  then  only 
be  necessary  to  proceed  on  the  lines  already  laid  down  in  the  sections  dealing 
with  the  isolation  of  organisms  and  with  the  examination  of  water.  It  is 
always  well  to  sow  both  agar  and  gelatin  plates  since  the  latter  generally 
liquefy  in  a  short  space  of  time. 

In  carrying  out  these  experiments  with  air  some  form  of  aspirator  is 
necessary.  For  choice,  a  water  aspirator  would  be  used  such  as  is  to  be 
found  in  chemical  laboratories.  With  the  aid  of  this  apparatus  the  volume 
of  air  aspirated  can  be  very  accurately  measured.  An  ordinary  water  exhaust 
pump  can  also  be  used.  In  this  case  it  will  of  course  be  necessary  to  interpose 
between  the  liquid  through  which  the  air  is  to  bubble  and  the  pump  a  gaso- 
meter which  will  record  the  volume  of  air  aspirated.  [A  still  more  simple 
and  quite  satisfactory  method  consists  in  using  a  large  glass  barrel  fitted  with 
a  tap  below  and  stoppered  above  with  an  india-rubber  plug  through  which  a 
narrow  piece  of  glass  tubing  is  passed,  such  as  is  used  in  operating  theatres 
for  storing  antiseptics.  This  vessel  can  be  graduated  once  for  all  by  pouring 
in  measured  volumes  of  water  and  marking  the  level  on  the  glass  with  a 
carburundum  pencil.  If  it  is  required  to  aspirate,  say,  10  litres  of  air,  the 
vessel  is  filled  with  water  up  to  the  10  litre  mark,  the  apparatus  is  then 
attached  to  the  small  glass  tube  above  and  by  regulating  the  flow  of  water 
from  the  tap  below  the  air  can  be  aspirated  at  any  speed  which  is  considered 
desirable.  For  the  examination  of  air  in  places  where  it  is  inconvenient  to 


FILTRATION  METHODS 


865 


use  any  large  piece  of  apparatus  Andrewes  uses  a  large  metal  aspirating  syringe 
of  known  capacity.  By  means  of  a  side  tap  the  aspirated  air  can  be  expelled 
without  disconnecting  the  syringe  from  the  filter  tube.] 

Whatever  the  form  of  aspirator  used  the  air  should  always  be  aspirated 
slowly  and  regularly  so  that  the  bubbles  burst  one  by  one  in  the  liquid  through 
which  it  is  passed.  There  are  various  other  pieces  of  apparatus  which  can 
be  used  for  the  same  purpose. 

A.  Methods  based  upon  filtration. 

1.  Filtration  through  insoluble  substances.    1.  Petri's  method.— Take  a 

piece  of  glass  tubing  about  15  mm.  in  diameter  and  10  cm.  long  and  at  each 

end  arrange  a  pair  of  wire  gauze  plugs  (Bp  B2,  B3,  B4,  fig.  412) 

leaving  a  space  of  about  3  cm.  between  eaclTpair  (C15  C2)  and 

fill  these  two  spaces  with  very  fine  sand  previously  heated  to 

redness.     Plug  the  two  ends  of  the  glass  tube  with  wool  and 

sterilize  the  apparatus  in  an  hot  air  sterilizer.     When  it  has 

cooled  replace  one  of  the  wool  plugs  with  a  sterile  perforated 

india-rubber  bung,  D,  through  which  a  piece  of  glass  tubing, 

F,  plugged  with  wool  is  passed.     To  use  the  apparatus,  attach 

the  end  of  the  small  glass  tube,  F,  to  an  aspirator,  take  the 

wool  plug  out  of  the  other  end  and  slowly  aspirate  100  litres 

of  air.     When  the  aspiration  is  completed  the  sand  is  mixed 

with  sterile  gelatin   and  a  number  of   plates  poured.     The 

method  is  complicated  and  of  little  use  in  practice. 

2.  Frankland's   method. — The   tubes  are   similar  to  those 
used  by  Petri  but  glass  wool  or  asbestos  is  substituted  for 
the  sand  :   this  does  away  with  the  necessity  for  the  metal 
gauze.     After  the  air  has  been  aspirated,  the  filtering  medium 
is  shaken  up  in  a  known  quantity  of  broth  and  the  tatter  is 
then  used  for  sowing  gelatin  plates.     This  method  though  very 
simple  is  not  exact  since  organisms  stick  to  the  glass  wool 
or  the  asbestos  and  do  not  become  suspended  in  the  broth. 

II.  Filtration  through  soluble  substances  (Pasteur). — By 
using  soluble  instead  of  insoluble  substances  the  distribution 
of  the  organisms  in  the  gelatin  is  made  more  certain  and 
their  enumeration  is  very  accurate.  Unfortunately  the 
method  is  not  applicable  when  the  atmosphere  contains  much  Petri'l' sand  mter 

moisture  since  in  that  case  the  filtering  substances  become   !?r  fch®  examina- 

.•         ,   ,.  ,         ,  5..  tion  of  air. 

moist,  deliquesce  and  no  longer  act  as  a  filter. 

Sulphate  of  sodium  is  ordinarily  used  as  the  filtering  medium.  The  salt 
is  fused  in  an  iron  vessel,  powdered  and  sifted  and  then  introduced  into  a 
glass  tube  of  the  shape  shown  in  fig.  413.  One  end  of  the  tube  is  plugged 


FIG.  413.— Glass  tube  for  soluble  filters. 


with  wool  and  beyond  this  is  a  constriction  against  which  rests  a  small  piece 
of  asbestos  and  then  powdered  sodium  sulphate  to  a  depth  of  about  8  cm., 
the  other  end  of  the  tube  is  drawn  out  and  sealed  in  the  flame.  The  apparatus 
is  sterilized  in  the  hot  air  sterilizer.  To  use  it,  the  powder  is  shaken  down 
against  the  asbestos  plug  by  gently  tapping  the  tube,  the  pointed  end  of  the 
tube  is  broken  off  and  the  other,  plugged,  end  connected  to  an  aspirator. 

3i 


866       THE   BACTERIOLOGICAL   EXAMINATION   OF   AIR 


When  the  desired  volume  of  air  has  been  aspirated  the  powdered  sodium 
sulphate  is  dissolved  in  a  known  volume  of  broth  and  plates  are  sown  with 
measured  quantities  of  the  liquid.  As  a  control  the  asbestos  plug  is  trans- 
ferred with  sterile  forceps  to  a  tube  of  broth  and  this  of 
course  should  remain  sterile. 

[III.  Andrewes'  method. — The  filtering  medium  consists 
of  a  mixture  of  glass  wool  (3-4  parts)  and  cane  sugar 
(1  part).  A  straight  piece  of  glass-tubing  is  used  without 
any  constriction  and  the  medium  rammed  in  fairly  tightly. 
After  plugging  the  ends  with  wool  the  apparatus  is  steril- 
ized at  120°  C.  and  after  aspirating  the  air  through  it  the 
mixture  is  pushed  out  with  a  sterile  glass  rod  into  a  plate 
of  melted  sterile  gelatin.] 

B.  Methods  based  upon  bubbling  the  air  through 

liquids. 

I.  Method  of  Straus  and  Wurtz. — The  apparatus  consists 
of  a  glass  cylinder  with  an  appendix  at  its  lower  end  filled 
with  10  c.c.  of  liquefied  gelatin  the  surface  of  which  is 
covered  with  a  few  drops  of  oil. 

The  upper  part  of  the  cylinder  is  furnished  (1)  with  a 
lateral  tubulure  plugged  with  wool  and  (2)  with  a  central 
J  ground  glass  opening  which  is  hermetically  closed  with  a 

"^  glass  tube — reaching  below  to  the  bottom  of  the  gelatin 

in  the  appendage  and  above  projecting  beyond  the  cylinder 
—which  is  plugged  with  wool.  The  apparatus  is  sterilized 
in  the  autoclave.  When  required  for  use,  the  lower  part 
of  the  cylinder  is  placed  in  water  at  about  10°  0.  to  liquefy 
the  gelatin,  the  lateral  tubulure  is  attached  to  an  aspirator 

and  the   wool   plug   removed.     The    as- 
pirated  air  passes   through   the   central 

tube  B  and  bubbles  through  the  gelatin 

in  which  it  deposits  the  organisms  sus- 
pended in  it.     (The  layer  of  oil  prevents 

the  gelatin  frothing.)     When  10  litres  of 

air  have  been  aspirated  the  aspirator  is 

disconnected,  and  air  is  gently  blown  in 

through  the  lateral  tubulure,  thus  driving 

the  gelatin  up  into  the  central  tube.    This 

operation  is  repeated  several  times  in  order 

to  thoroughly  wash   the  tube.     Finally, 

plates  are  poured  with  the  gelatin.     [The 

apparatus   can  be  used    for   the   simple 

enumeration  of  organisms,  the  gelatin  in 

this  case  being  run  over  the  sides  of  the 

cylinder  after  the  fashion  of  an  Esmarch's 

roll  tube.] 

This  apparatus  is  very  convenient  but  many  organisms  are  arrested  in  the  delivery 
tube  which  is  very  long  and  has  an  irregular  surface,  so  that  the  results  are  not  very 
accurate ;  moreover  the  apparatus  is  only  available  for  small  volumes  of  air. 

II.  Miguel's  method. — The  apparatus  consists  of  a  Pasteur  flask  with  two 
lateral  tubulures  attached  to  opposite  sides  of  its  upper  part  and  with  a 
central  tube  dipping  to  the  bottom.  A  ground  glass  cap  closes  the  central 
tube  :  one  of  the  lateral  tubulures  is  plugged  with  wool  and  the  other,  used 


FIG.  414.— Straus  and 
Wurtz'  apparatus. 


FIG.  415. — Miquel's  apparatus. 


LAVERAN'S  METHOD 


867 


for  distributing  the  medium  at  the  end  of  the  experiment,  is  drawn  out  and 
sealed  in  the  flame. 

Thirty  c.c.  of  water  are  poured  into  the  flask  and  then  sterilized  in  the 
autoclave.  To  use  the  apparatus  connect  the  plugged  tubulure  with  an 
aspirator,  remove  the  glass  cap  from  the  central  tube,  and  start  the  aspirator : 
the  air  now  bubbles  through  the  water  in  the  flask  and  when  sufficient  air 
has  been  drawn  through,  wash  out  the  central  tube  several  times  by  blowing 
gently  through  the  lateral  tubulure.  Then  break  off  the  sealed  end  of  the 
other  lateral  tube  and  distribute  the  contents  of  the  flask  into  a  number 
(30  or  40)  of  flasks  containing  broth  ;  these  flasks  are  then  incubated.  The 
method  is  not  accurate  because  the  mere  dipping  of  the  tube  into  the  liquid 
is  insufficient  to  entrap  all  the  organisms  and  conse- 
quently many  escape  detection. 

III.  Laveran's  method.  Method  recommended. 
Laveran  employs  a  piece  of  apparatus  which  while  <  M 
giving  very  accurate  results,  is  simple  in  construction 
and  not  easily  broken.  Two  glass  tubes  closed  below 
are  connected  together  at  the  junction  of  the  middle 
and  upper  thirds  by  an  horizontal  tube.  Each  of  the 
vertical  tubes  is  plugged  at  its  upper  end  with  an  india- 
rubber  stopper  through  which  a  pipette  reaching  to  the 
bottom  of  the  tube  is  passed.  One  of  the  tubes  has  a 
10  c.c.  mark  on  the  glass  and  one  of  the  pipettes  is 
graduated  in  tenths  of  a  cubic  centimetre.  The  upper 
end  of  each  pipette  is  plugged  with  wool.  10  c.c.  of  a 
1  per  cent,  solution  of  sugar  in  water  are  poured  into 
the  graduated  tube.  The  apparatus  is  then  autoclaved. 

For  use,  remove  the  wool  plug  from  the  pipette 
dipping  into  the  sugar  solution  and  connect  the  other 
with  an  aspirator.  The  aspirated  air  bubbles  through 
the  solution,  passes  into  the  first  tube  through  the  horizontal  connecting 
tube,  descends  in  the  other  limb  and  escapes  through  the  pipette  connected 
with  the  aspirator.  A  very  large  volume  of  air  can  thus  be  aspirated. 

When  sufficient  air  has  bubbled  through  gently  aspirate  the  sugar  solution 
into  the  entry  pipette  to  wash  it,  then  run  the  fluid  into  the  second  tube 
and  so  into  the  second  pipette,  and  repeat  this  several  times  in  order  to  collect 
all  the  organisms  which  have  been  deposited  on  the  glass.  It  only  remains 
now  to  remove  the  liquid  by  means  of  the  graduated  pipette  and  to  distribute 
it  into  different  culture  media  (gelatin  and  agar  plates,  etc.). 

Suppose,  for  example,  that  200  litres  of  air  have  been  aspirated  and  twelve  colonies 
have  developed  on  a  gelatin  plate  sown  with  one  cubic  centimetre  of  the  sugar 
solution,  it  follows  that : 

200  litres  of  air  contain  12  x  10  aerobic  organisms. 

1  cubic  metre  therefore  contains  12  x  10  x  5  aerobic  organisms. 

The  advantages  of  the  method  are  that  it  is  available  for  large  volumes  of 
air  and  provides  plenty  of  material  for  sowing  cultures  :  thus  it  fulfils  the 
requirements  of  special  investigations  for  the  detection  of  pathogenic  micro- 
organisms.1 

1  For  details  reference  must  be  made  to  the  chapters  devoted  to  the  consideration  of 
the  various  pathogenic  micro-organisms.  For  the  detection  of  the  tubercle  bacillus  it 
will,  of  course,  be  necessary  to  inoculate  animals. 


FIG.  416  — 
Laveran's  tube. 


INDEX. 


Abbe's  apochromatic  objectives,  115. 

-  sine  law  for  aplanatism,  110. 
Abe's  agar,  639. 

Abscesses,  collection  of  pus  from,  197. 

—  spontaneous,  in  animals,  159. 
Absorption  of  agglutinins,  method  of,  436. 

-  —   (aertrycke  bacillus),  441. 

—  —  —   (paratyphoid  A  bacillus),  427. 
(        —  B      —      ),  436. 

-  (typhoid  bacillus),  389. 

.  —  —  oxygen  from  culture  media,  89. 
Acanthia  lectularia  and  spirochsetosis,  711. 
Acari,  spontaneous  infection  of  rabbits 

with,  159. 
Acetic  perchloride  as  hardening  reagent, 

189. 

-  violet,  148. 
Acetone  alcohol,  143. 

Achalme's  rheumatism  bacillus,  569,  570. 

—  blood-broth  medium,  34. 
Achorion  arloingi,  692. 

—  quinckeanum,  692. 

—  schoenleini,  690. 
Achromatism,  114. 
Acid  alcohol,  346. 
Acid  dyes,  136. 
Acid-fast  bacilli,  345. 

-  in  urine,  343. 
butter,  338. 

Acne  varioliforme,  766. 
Acquired  immunity,  221. 
Actino-bacillosis,  661. 
Actinobacillus  lignieresi,  661. 
Actino-congestine,  224. 
Actinomyces  bovis,  656. 
Actinomycosis,  656,  665. 

-  parasites  of,  660. 
Actinomycotic  mycetoma,  665. 

Action  of  oxygen  on  potassium  pyrogallate, 
89. 

-  vitality  of  micro-organisms,  75. 
Active  immunity,  221. 

Acute  anterior  poliomyelitis,  virus  of,  844. 
Adelea  (genus),  766. 
Agar-agar,  42. 
Agar,  Abe's,  639. 

—  ascitic,  53. 

-  (Ruediger's),  585. 

-  blood,  53. 


Agar,  gelatin,  43,  44. 

-  glucose-glycerin,  44. 

—  glycerin,  44,  693. 

—  Heiman's,  639. 

-  Hesse's  (tubercle  bacillus),  317. 

—  Krai's,  639. 

-  Leipschutz's,  639. 

—  litmus,  57. 

—  media,  42. 

—  Malm's,  44. 

—  Nasstikoff's,  639. 

—  Sabouraud's,  673,  681. 

-  Salomonsen's,  44. 

—  serum,  53. 

-  Steinschneider's,  640. 

-  Tochtermann's,  317. 

—  Wertheim's,  638. 

—  Wildbolz's  agar,  639. 
Agglomeration  (T.  brucei),  812. 

-  (T.  lewisi),  807. 
Agglutinins,  225. 

-  heterologous,  389. 

—  homologous,  389. 

—  primary,  389. 

—  secondary,,  389. 

-  specific,  389. 
Agglutination,  mechanism  of,  226. 

-  (measurement  of  titre),  388. 

—  of  micro-organisms   (see  the  several 
chapter  headings). 

Agressins,  222  (footnote). 

Aertrycke  bacillus,  438. 

Aerobic  micro-organisms,  definition  of,  28. 

-  —   isolation  of,  76. 

—  —   methods  of  sowing  and  cultivat- 
ing, 67. 

^Estivo-autumnal  fever,  780. 

Air  (bacteriological  examination),  862. 

Albuminous  media,  45. 

Alcohol  ether,  141. 

Alcoholic  staining  solutions,  137. 

Aleppo  boil,  802. 

Alexin,  229. 

Alopecia  areata,  692. 

Amboceptor,  229. 

American  diphtheria  bacillus,  257,  259. 

-  relapsing  fever,  712. 

-  trypanosomiasis,  823. 
Amoeba  buccalis,  745. 


INDEX 


869 


Amceba  coli,  747. 

—  dysenterica,  747. 

—  histolytica,  748. 

—  pelaginia,  747. 

—  princeps,  745. 

—  urogenitalis,  745. 

—  vaginalis,  745. 
Amoebic  dysentery,  356. 
Amoeboid  parasite  of  malaria,  774. 
Anaemia  of  horses,  virus  of,  842. 
Anaerobic  micro-organisms,  action  of  oxy- 
gen on,  87. 

-  cultivation  of,  in  liquid  media,  92. 

-  —  in  solid  media,  99. 
-  —  on  solid  media,  100. 

-  growth  of,  in  presence  of  oxygen, 
87. 

—  —  isolation  of,  87. 

(Plate  method),  101. 

(Tube  method),  103. 

-  in  gangrene,  569. 
Anaphylaxis,  224. 
Anasarca  of  the  horse,  607. 

Andrewes'    method    (examination   of  air), 

866. 
Andrewes    and    Border's    classification    of 

streptococci,  601. 
Angers  vibrio,  492. 
Angina,  Vincent's,  574. 
"  Angines  sableuses,"  631. 
Angular  aperture,  112. 
Aniline  dyes  (see  Stains),  136. 

—  oil  water,  139. 
Animal  inoculation,  156. 
Animals,  diphtheria  bacillus  in,  246. 
Anopheles  mosquito,  776,  777. 
Ante  mortem  (agonic)  infections,  393. 
Anthrax,  517. 

—  examination  of  carcases  dead  of,  534. 

—  symptomatic,  552. 

Antiformin    (isolation    Tubercle    bacillus), 

341 

Antigen,  229,  235. 
Antiseptics,  sterilization  by,  26. 
Antitoxin  (Bacillus  botulinus),  551. 

—  (diphtheria),  265. 

—  —  anaphylaxis  following,  224. 

-  dried,  266. 

—  —   properties  of,  266. 

-  unit  of,  268. 

-  in  milk,  266. 

—  (Tetanus),  545. 
Antitoxins,  224. 
Antityphoid  vaccination,  380. 

-  effects  of,  382. 

-  Wright-Leishman  method,  381. 

-  Besredka's  method.  382. 
Antibody,  229. 
Anti-colilysin,  399. 
Anti-scarlatinal  vaccination,  604. 
Antityphoid  serum  (Besredka's),  384. 

-  (Chantemesse's),  383. 
Antitypholytic  serum,  379. 
Ants  and  plague,  462. 
Apertometer,  112. 

Apes,  natural  plague  in,  460. 
Aphthous  fever,  virus  of,  838. 


Aplanatic  image,  110. 

Aplanatism,  sine  condition  for,  110. 

Apochromatism,  114. 

Apochromatic  objectives,  115. 

Appearances  presented  by  micro-organisms 

in  culture,  73. 
Aqueous  humour,  collection  of,  197. 

—  solutions  of  dyes,  137. 

Argas  reflexus  and  Sp.  marckouxi,  718. 

—  persicus  and  Sp.  marchouxi,  718. 
Arloing's  vaccines  for  quarter  ill,  556. 

—  serum  for  quarter  ill,  559. 

—  method    of   immunization   of   cattle 
against  the  tubercle  bacillus,  333. 

—  homogeneous      cultures      (Tubercle 
bacillus),  335. 

Aronson's  antistreptococcal  serum,  607. 
d'Arsonval's  incubator,  63. 

electric,  65. 

Arterial  inoculation,  173. 
Arthrospore,  145. 
Artichoke  as  culture  medium,  375. 
Ascitic  fluid,  collection  of,  198. 
Ascophora  nigricans,  678. 
Asiatic  cholera,  488. 
Aspergillary  mycetoma,  665. 
Aspergillus  (genus),  695. 

—  bouffardi,  665. 

—  concentricus,  700. 

—  fumigatus,  695. 

—  glaucus,  695. 

—  herbariorum,  695. 

—  lepidophyton,  700. 

—  malignus,  695. 
— •  nidulans,  699. 

—  niger,  699. 

—  pictor,  698. 

—  repens,  695. 
Aspiration,  filtration  by,  19. 
Aspirator,  Potain's,  82. 

Asses,  collection  of  blood  from,  193. 

—  handling  of,  164. 

—  Nagana  in,  811. 
Asylum  dysentery,  356. 
Atelosaccharomyces  busse-buschki,  706. 
Auer  gas  burner,  118. 
Autoclaves,  9. 

Avian  pasteurella,  447. 

—  tubercle  bacillus,  291. 

—  tuberculosis,  296. 

Babes'  granules,  252. 

—  incubator,  63. 

—  temperature  regulator,  59. 
Babesia,  787  footnote. 

—  bigemina,  787. 

—  ovis,  791. 

-  parva,  793  footnote. 
Bacillary  dysentery,  356. 

-  pseudo-tuberculoses,  347. 
Bacille  septique  aerobie,  578. 
Bacillus  aerobicus  sepis,  578. 

—  aerogenes  capsulatus,  569. 

—  anthracis,  517. 

—  —   brevigemmans,  524. 

—  asciformis,  693. 

—  botulinus,  549. 


870 


INDEX 


Bacillus  chauvcei,  552. 

—  cholerce  suis,  438. 

—  coli,  393. 

—  diphtheria,  245. 

—  entericce  febris,  366. 

—  enteritidis  aertrycke,  438. 

—  —  gaertner,  442. 

-  pseudo-gaertner,  444. 

—  dysenterice  epidemicce,  356. 
El  Tor  No.  1,  365. 

—  F,  437. 

-  fragilis,  573. 

—  funduliformis,  572. 

-  fusiformis,  574. 

—  hc&moglobinophilus  canis,  510. 

—  hastilis,  574. 

—  icteroides,  445. 

-  influenzce,  504. 

-  lactis  aerogenes,  415. 

-  leprce,  348. 

—  maligni  oedematis,  561. 

—  mallei,  480. 

—  paratyphosus  A,  423. 
B,  431. 

—  perfringens,  569. 

-  pertussis  Eppendorf,  510. 

—  pestis,  460. 

—  phlegmonis  emphysematosce,  569. 
posciloides,  571. 

—  pseudo-diphtherice,  247,  273. 

-  pseudo-oedema,  571. 

-  pseudo-tuberculosis    rodentium,     160, 
474. 

—  pyocyaneus,  276. 

—  ramosus,  571. 

—  serpens,  572. 

-  smegmce,  346. 

—  suipestifer,  438. 

-  tetam,  536. 

—  thetoides,  572. 

—  tuberculosis,  289. 

-  typfo'  murium,  444. 

—  typhosus,  366. 

Bacillus  of  acute  contagious  conjunctivitis, 
510. 

-  blue  pus,  276. 

-  coscoroba  swan  disease,  452. 
•  — -   Danysz,  444. 

-  distemper,  457. 
(M'Gowan),  459. 

-  duck  cholera,  452. 

-  —   epizootic   dysentery   in    turkeys, 
452. 

—   ferret  septicaemia,  453. 

—  — -  fowl  cholera,  447. 

—  enteritis,  452. 

Friedlander,  415. 

glanders,  480. 

-  green  diarrhoea,  400. 

• —  —  haemorrhagic  septicaemia  of  ducks 
and  fowls,  453. 

-  hog  cholera,  438. 

-  Karlinski,  354  footnote. 

—  —  malignant  oedema,  561. 

-  mouse  septicaemia,  288. 
ozoena,  419. 

-  pseudo-tuberculosis,  160,  474. 


Bacillus  of  psittacosis,  445. 

-  quarter  ill,  552. 

-  rabbit  septicaemia,  453. 
-  —   rhinoscleroma,  418. 

seborrhcea  oleosa,  692. 

-  soft  sore,  513. 

—  —   sub-acute  conjunctivitis,  511. 

—  —   swine  erysipelas,  283. 

-  symptomatic  anthrax,  552. 

-  Tavel,  306  footnote. 

—  —   the  peladic  utricle  in  alopecia,  692. 

-  verruga  peruana,  346. 

—  —   whooping  cough,  511. 

—  —  wood  pigeon  disease,  452. 
Bacteria,  136. 

—  vitality  of  phagocyted,  222. 
Bactericidal  substances,  origin  of,  223. 

—  serums,  227. 

Bacteriolysis,  mechanism  of,  228. 
Bacteriolytic  serums,  preparation  of,  236. 
Bacteriotropins,  239. 

Bacterium  murisepticurn,  288. 

—  photometricum,  flagella  of,  148. 
Balantidium  coli,  356,  830. 

—  minutum,  831. 
Balbiania,  756. 

—  gigantea,  758. 

—  mucosa,  758. 

—  siamensis,  758. 
Bald  ringworm,  688. 
Balori,  814. 
Barbeiro,  822. 
Barbone,  455. 

Barrel-shaped  distortion   of  microscopical 

image,  109. 

Barrouxia  (genus),  766. 
Barsiekow's  culture  medium,  57. 
Basic  dyes,  136. 
Bassett-Smith's     vaccine     (Mediterranean 

fever),  478. 

Bats,  Haematozoon  of,  781. 
Baumgarten's  stain  (Leprosy  bacillus),  350. 
Bayon's  culture  medium  (Leprosy),  353. 
Beef  broth  culture  medium,  30. 
Bee  disease  (Mucor  mucedo),  677. 

—  —  (Nosema  apis),  753. 
Beggiatoa  roseopersinica,  flagella  of,  148. 
von    Behring's    method    of    standardizing 

diphtheria  antitoxin,  267. 

-  immunizing  cattle  against 

tuberculosis,  330. 

Beraneck's  tuberculous  acido-toxin,  329. 
Berkefeld  filter,  15. 

-  —   cleansing  of,  18. 
Bernard's  bit,  164. 
Bertarelli's  stain  (Amoebae),  749. 

(Coccidia),  761. 

Bertarelli,  Volpino  and  Bovero's  stain  (T. 

pallidum),  730. 
Besredka's  antistreptococcus  serum,  609. 

-  antityphoid  serum,  384. 

-  sensitized  vaccine  in  cholera,  498. 

dysentery,  362. 

-  enteric  fever,  383. 

-  plague,  470. 

—  —   streptococcal     infections. 
605. 


INDEX 


871 


Besredka's  typhoid  endotoxin,  379. 
Besson's   method   of  preparing   malignant 
cedema  toxin,  566. 

-  isolating    the     typhoid     bacillus 
from  water,  405. 

Besson    and    Pourrat's    method    of   intra- 

pleural  inoculation,  179. 
Benignetti  and  Gino's  method  for  staining 

flagella,  152. 
Bezaneon  and  Griffon's  blood-agar.  53. 

-  (Tubercle  bacillus),  317. 

—  —   egg  medium  (Tubercle  bacillus), 
316. 

Bichromate  solution,  131. 
Bile  as  a  culture  medium,  410. 

-  for  cholera,  501. 

-  for  the  typhoid  bacillus,  372. 

-  for  the  tubercle  bacillus,  318. 
Biliary  fever  of  dogs,  791. 

Biliary  passages,  inoculation  into  the, 
177. 

Biochemical  reactions  (see  the  several  chap- 
ter headings). 

Biological  properties  (see  the  several  chap- 
ter headings). 

Birds,  bleeding  of,  195. 

-  coccidiosis  in,  764. 

—  haematozoa  in,  781. 

-  handling  of,  165. 

—  in tra- venous  inoculation  of,  173. 

—  post  mortem  examination  of.  188. 

—  tuberculosis  in,  296. 

-  trypanosomes  in,  823. 
Bird  plague,  virus  of,  839. 
Black  mycetoma,  665. 

—  quarter,  552. 

—  tongue,  678,  706. 
Blastomyces  dermatitis,  706. 
Blastomyces  and  cancer,  707. 
Blastomycetes,  701. 
Blastomycetic  dermatitis,  706. 
Blastomycosis,  706. 

Blood  as  a  culture  medium,  36. 

—  -agar,  53,  639. 

-  -broth,  34,  638. 

—  collection     of,      for     bacteriological 
examination,  192. 

—  —   from  a  vein  at  the  bend  of  the 
elbow,  193. 

-  (Nseggerath       and       Staehelin's 
method— Syphilis),  733. 

-  —   (Nattan-Larrier    and    Bergeron's 
method — Syphilis),  733. 

-  (Ravaut  and  Ponselle's  method — 
Syphilis),  733. 

-  defibrination  of,  36. 

—  -films,  preparation  of,  204. 

-  —   staining  of,  206. 

—  —  —  for  gram-negative  organisms, 
209. 

-  unstained  preparations,  203. 
Blue  pus,  276. 

-   tongue,  839. 
Bodo  urinarius,  827. 
Boehmer's  hsematoxylin,  218. 
Bcettcher's  hollow-ground  slide,  134. 
Bohr's  temperature  regulator,  60. 


Bombicci's  dish  (Isolation  of  anaerobes), 

101. 
!    Bone  marrow,  bacteriological  examination 

of,  187. 

Bonome's  streptococcus.  610. 
Boophilus  annulatus  and  P.  bigeminum,  787. 

—  decoloratus,  787. 

-  decoloratus  and  S.  theileri,  719. 

-  dugesi  and  P.  bigeminum,  787. 
Borax  blue,  139,  771. 

-   -methylene-blue,  514. 
Bordet-Gengou  reaction,  232. 
Bordet-Gengou's     bacillus     of     whooping 

cough,  511. 
Borrel's  blue,  139,  772. 

—  broyeur,  170. 

-  stain  (Sporozoa),  761. 

Borrel  and  Burnett's  stain  (T.  pallidum), 
728. 

Bottle  for  cultivation  of  anaerobic  micro- 
organisms, 94. 

Botulism,  549.     . 

Bougie,  porous  porcelain  (see  Filters),  14. 

Bovine  farcy,  480,  667. 

—  pasteurellosis,  455. 

—  piroplasmosis,  787. 

—  tubercle  bacillus,  289. 

—  tuberculosis,  294. 
Bovo-vaccin,  von  Behring's,  330. 
Bowhill's  stain  for  flagella,  152. 
Bradford  disease,  517. 
Branching  bacilli,  245  footnote. 

-  diphtheria  bacilli,  251. 

—  tubercle  bacilli,  313. 
Bread  as  a  culture  medium,  56. 
Breeding  of  small  animals,  159. 
Brightness  of  image,  114. 
Brilliant-green  media,  411. 
Brisou  coccus,  249. 

Broth,  30. 

—  arsenical,  375. 

-  for  diphtheria,  253. 
Broyeur,  Borrel's,  170. 

Brush  rats,  natural  plague  in,  461. 
Bubonic  plague,  460. 

Buchner's  method  (Cultivation  of  anae- 
robes), 95. 

-  gelatin,  41. 

Buffaloes,  pasteurellosis  in,  455. 

—  plague  in,  461. 

—  sarcosporidiosis  in,  758. 
Bugs  and  kala  azar,  799,  801. 

-  and  oriental  sore,  802. 

-  and  plague,  462. 

—  and  spirochsetosis,  711. 

-  and  trypanosomiasis,  822. 

Bug  of  Mianeh  (Argas  persicus)  and  spiro- 
chaetosis,  712. 

Bulb  pipette,  22. 

Bulloch's  apparatus  (Cultivation  of  anae- 
robes), 96,  100,  102. 

Bumm's  serum  medium  (Gonococcus),  640. 

Bunge's  stain  for  flagella,  151. 

Burri's  method  (Isolation  of  micro-organ- 
isms), 83. 

Bursattee,  674. 

Butter,  acid-fast  bacilli  in,  338. 


872 


INDEX 


Caffeine  media,  375,  408. 
Cages  for  experimental  animals,  157. 
Calmette's  bile  medium  (Tubercle  bacillus), 
318. 

—  ophthalmo-reaction    in    tuberculosis, 
327. 

—  tuberculin,  328. 

Calmette  and  Salimbeni's  plague  vaccine, 

470. 
Cambier's  method  (Isolation  of  the  Typhoid 

bacillus),  406. 

-  (Motility     of     micro-organisms), 
154. 

Cambridge  "  rocking  "  microtome,  211. 
Camels  and  surra,  814. 
Camera  lucida  for  measuring  magnification, 
121. 

-  microscopical  objects,  122. 
Camphor  as  an  antiseptic,  26. 

Camus'    method   for   collection    of  serum, 

197. 

Canada  balsam,  142. 
Cancer,  filtrable  viruses  in,  840. 

-  Saccharomyces  and,  707. 

-  spirochsetes  in,  735. 
Canguary,  822. 

Canine  leishmaniosis,  801. 

-  pasteurellosis,  456. 

—  piroplasmosis,  791. 

—  typhoid,  457. 

Cantacuzene's  stain  (T.  pallidum),  717. 
Capaldi's  egg  medium  (Tubercle  bacillus), 

316. 

Caps,  india-rubber,  29. 
Capsule,  Wright's  blood-collecting,  192. 
Capsules,  staining  of,  147. 
Capybara  and  Mai  de  Caderas,  814. 
Carates,  698. 
Carbohydrate  media,  34. 

-  sterilization  of,  by  filtration,  35. 
Carbolic   acid,   use   of,   in  preparation   of 

vaccines,  27.  . 
Carbolic -agar,  407. 
broth,  402. 

-  -gelatin,  402. 
Carbol-crystal-violet,  138. 

-  -fuchsin,  138. 

-  dilute,  138. 

-  -gentian-violet,  138. 
-methylene-blue  (Kiihne's),  138. 

-  -thionin,  138. 
Carbonated  broth,  35. 

Carbonic  anhydride,  generation  of,  89. 

-  sterilization  by,  11. 
Carceag,  791. 

Cardiac  puncture  in  guinea-pigs,  194. 

-  rabbits,  195. 
Carmine  (Orth's),  218. 
Carmine,  alcohol  (Orth's),  218. 
Carnot  and  Gamier' s  tube,  155. 
Carotid  artery,  inoculation  into,  174. 
Carp,  microsporidiosis  in,  754. 

—  myxosporidiosis  in,  756. 
Carriers  in  cholera,  488. 

—  enteric  fever,  367. 

-  (complement    fixation    reac- 
tion), 390. 


Carriers  of  meningococcal  infections,  645. 

—  in  paratyphoid  A  fever,  423. 

—  B  fever. 
Carrot,  infusion  of,  37. 

Castellani's  absorption  of  agglutinins  (see 

Absorption  of  agglutinins),  427. 
Castor  oil,  refractive  index  of,  119. 
Catarrhal  fever  of  sheep,  839. 
Catheterization  of  the  oesophagus,  181. 
Cats  as  experimental  animals,  157. 

-  coccidiosis  in,  764. 

—  diphtheria  in,  246. 

—  distemper  in,  457. 

—  handling  of,  164. 

—  leishmaniosis  in,  802. 

—  natural  plague  in,  461. 
— •   ringworm  in,  687. 

—  tuberculosis  in,  296. 

Cattle  as  experimental  animals,  156. 

—  collection  of  blood  from,  48,  193. 

—  East  coast  fever  of,  793. 

—  Gaertner's  bacillus  in,  442. 

—  handling  of,  165. 

—  in tra- venous  inoculation  of,  173. 

-  pasteurellosis  of,  455. 

-  plague  in,  461. 

—  red  water  of,  787. 

-  Rhodesian  fever  of,  793. 

—  spirocha3tosis  of,  719. 

-  tropical  piroplasmosis  of,  793. 

-  trypanosomiasis  of  (Souma),  814. 

—  tuberculosis  of,  294. 
Cattle  plague,  virus  of,  839. 

Cedar  wood  oil,  refractive  index  of,  119. 
Cell  inclusions  in  rabies,  841. 

—  —   swine  fever,  843. 
trachoma,  843. 

Central  nervous   system,   examination   of, 

187. 
Ceratomi/xa  appendiculata,  756. 

—  incequalis,  756. 

—  linospora,  756. 
C&ratophyllus  fasciatus  on  rats,  461. 

—  and  Trypanosoma  leivisi,  805. 
Cercomonas  hominis,  825. 

—  intestinalis,  825. 

—  termo,  826. 

Cerebro-spinal  fluid  in   general  paralysis, 

738. 

Cerrito's  stain  for  flagella,  151. 
Chagas'  disease,  822. 
Chamberland's  autoclave,  9. 

—  bougie  (filtering),  15. 

—  filtering  apparatus,  24. 

—  flask,  46. 

Chamberland  and  Roux's  asporogenic  an- 
thrax bacillus,  527. 
Chancel's  temperature  regulator,  59. 
Chantemesse's  antityphoid  serum,  383. 

-  hot  air  sterilizer,  5. 

—  method   of  isolating   aerobic   micro- 
organisms, 82. 

-  methods    of    isolating    the    typhoid 
bacillus,  85,  407,  412. 

-  typhoid  toxin,  377. 

Chantemesse  and  Widal's  method  of  isolat- 
ing the  typhoid  bacillus,  402,  407. 


INDEX 


873 


Chemiotaxis,  222. 

Chenzinsky's  stain  for  blood,  210. 

Chicken  broth,  32. 

Chilodon  dentatus,  831. 

China  green  medium,  410. 

—  ink  in  detection  of  T.  pallidum,  728. 
Chlamydozoa,  843. 

Chloroform  as  an  antiseptic,  27,  46. 
Cholera  vibrio,  488. 

—  intestinal,.  489. 

Choleraic  peritonitis,  experimental,  489. 

—  septicaemia,  experimental,  489. 
Choquet's  gelatin,  41. 
Chromatic  aberration,  110,  114. 
Cibil's  extract,  34. 

Cimex  lectularius  and  Indian  kala  azar,  799. 

—  and  Mediterranean  kala  azar,  801. 

-  and  plague,  462. 

-  rotundatus  and  Indian  kala  azar,  799. 

—  —   and  Mediterranean  kala  azar,  801. 

-  and  oriental  sore,  802. 
Citrate  solution,  169,  804. 
Clado's  urinary  bacillus,  393. 
Classification  of  diphtheria  bacilli,  250. 

—  dysentery  bacilli,  357,  360. 

-  malarial  parasites,  780. 

-  streptococci,  596,  601. 

-  the  parasites  of  actinomycosis,  660. 

—  mycetoma,  666. 

-  the  tricophyta,  682. 

-  the  tripanosomidaa,  804. 
Claudius'  stain  for  films,  144,  209. 

—  sections,  219. 
Clavelization,  840. 

Coagulation  of  blood,  prevention  of,  36. 

-  serum,  51. 
Co-agglutinins  in  enteric  serums,  389. 

-  paratyphoid  A  fever,  426. 

-  experimental  aertrycke  serums,  441. 

-  meningococcal,  649. 
• —  —   paratyphoid  B,  435. 

Coal  gas  (Cultivation  of  anaerobic .  micro- 
organisms), 89. 
Cobbett's  bulb,  18,  19,  24,  45. 

—  classification    of    diphtheria    bacilli, 
250. 

—  medium  for  diagnosis  of  diphtheria, 
271. 

Coccidia  in  tumours,  766. 
Coccidiidea,  760. 
Coccidioidal  granuloma,  706. 
Coccidioides,  671. 

—  immitis,  671. 
Coccidiosis  in  man,  761. 

—  rabbits,  160. 
Coccidium,  760. 

—  avium,  764. 

—  bigeminum,  760,  764. 

—  cuniculi,  760. 

—  falciforme,  764. 

—  hominis,  761. 

—  jalinum,  764. 

-  oviforme,  160,  760. 

—  perforans,  761. 

—  pfeifferi,  764. 

—  proprium,  764. 

—  salamandrce,  764. 


Coccidium,  tenellum,  764. 

—  truncatum,  764  footnote. 
Coccoid  granules  in  spirochsetes,  712. 
Coccus  Brisou,  249. 

Cochin  China  diarrhoea,  827. 
Cockroaches  and  plague,  462. 
Cohn's  culture  medium,  38. 
Cold-blooded  vertebrata,   Haemogregarines 
in,  783. 

—  —  Trypanosomes  in,  824. 
Tuberculosis  in,  296. 

Colilysin,  398. 

Collection  of  material  for  bacteriological 
examination,  191. 

—  —  post  mortem,  185. 

—  —  from    suspected    cases    of    diph- 
theria, 270. 

-  syphilis,  732. 

—  water    for    bacteriological    examina- 
tion, 851. 

Collodion  sacs,  174. 
Coloured  media,  56. 
Colpoda  cucullus,  831. 
Coma,  110. 
Comma  bacillus,  488. 
Compensating  collar,  115. 

-  oculars,  115. 
Complement,  229. 

—  fixation  reaction,  232. 

—  —  —  practical  applications,  238. 

-  preparation  of,  235. 

—  titration  of,  235. 
Condenser,  Abbe's,  106. 

Condensers  for  dark-ground  illumination, 
125. 

Conditions  essential  to  growth  of  micro- 
organisms, 72. 

Congress  of  Hygiene,  1894,  265. 

Conorrhinus  megistus  and  Chagas'  disease, 
822. 

—  rubrofasciaius  and  Indian  kala  azar, 
800. 

Conradi's  brilliant-green  culture  medium, 
411. 

Conradi  and  Drigalski's  culture  medium, 
376?  407. 

Contacts  in  diphtheria,  246. 

Contagious  pneumonia  of  horses,  456. 

Contaminations,  definition  of,  76. 

Coplans'  medium  for  diagnosis  of  diph- 
theria, 271. 

Copper  cylinders  for  sterilizing  pipettes, 
etc.,  4. 

Cornet's  forceps,  131. 

Corpuscles  of  Cornalia,  752. 

Correction  of  lenses  for  achromatism,  115. 

Corrosive  sublimate  as  hardening  agent, 
189. 

Corynebacteria,  characteristics  of,  245  foot- 
note. 

Corynebacterium  diphtherice,  245  footnote. 

—  commune,  245  footnote. 

—  conjunctiva,  245  footnote. 

—  mallei,  245  footnote. 
Courbevoie  vibrio,  491,  492. 
Cover-glasses,  effect  of  thick,  119. 

—  cleaning  of,  130. 


874 


INDEX 


Cows,  contagious  maramitis  of,  613. 

—  diphtheria  in,  246. 

—  tuberculosis  in,  290,  294. 
Cow-pox,  virus  of,  840. 
Crescent  parasites  in  malaria,  775. 
Cresoidin  solution,  253. 

Croup,  245. 

-  diagnosis  of,  269. 
Cryptococcus,  701  footnote. 

—  degenerans,  707. 

—  dermatitis,  706. 

—  farcinosus,  706. 

—  gikhristi,  706. 

—  hominis,  706. 

-  linguae,  pilosce,  706. 

—  lithogenes,  706. 

-  tokishigei,  707. 
Ctenocephalus  canis  on  rats,  461. 
Ctenopsyllus  musculi  on  rats,  461. 
Culex  pipiens,  782. 

Cultivation  of  aerobic  micro-organisms,  67. 

—  anaerobic  micro-organisms,  92. 

— -  micro-organisms     (see     the     several 
chapter  headings). 

—  on  special  media  for  isolation,  85. 
Cultural  characteristics  of  micro-organisms 

(see  the  several  chapter  headings). 
Culture  media,  agar,  42. 

—  of  animal  tissues  and  fluids,  30. 

-  gelatin,  39. 

-  liquid,  30. 

-  litmus,  56. 

-  meat,  54. 

-  rabbit-blood  broth,  584. 

-  solid,  39. 

-  synthetic,  38. 

-  vegetable,  37,  55. 

-  removal  of  air  from,  87. 

-  sterilization  of,  9. 

—  —   by  filtration,  18. 

-  tray  for  sloping,  52. 

-  tubes,  sealing  of,  30. 

-  vessels,  29. 

Cultures,  macroscopical  characteristics  of, 
73. 

—  examination  of,  73. 

—  —  microscopically,  130. 

-  inoculation  of,  169,  170. 

—  isolation  of  organisms  by  stroke,  81. 

—  methods  of  storing,  75. 

—  pure,  76. 
Cupping,  193. 

Curtis'  stain  (Sacch.  tumefaciens),  705. 
Cuti-reaction,  von  Pirquet's  (Tuberculosis), 

327. 

Cyclospora,  766. 

Cyprinodont  fish,  Microsporidia  in,  754. 
Cystomonas  urinaria,  827. 
Cystospermium  villorum  intestinalium  canis, 

764. 

Cytase,  229. 
Cytotoxins,  353. 
Czermak's  apparatus  for  holding  animals, 

161. 

Danilewskya,  783. 

—  lacazei,  785. 


Danysz's  virus,  444. 
Dark-ground  illumination,  123. 
Davidson's  stain  (T.  pallidum),  729. 
Dean's  (G.)  diphtheria  toxin,  259. 
Dean's  (H.  R.)  technique  for  complement 

fixation  reaction,  428. 
Debove's  syringe,  167. 
Debrand's  apparatus  for  holding  animals, 

161. 

—  forceps,  131. 
Defibrination  of  blood,  36. 

"'  Definition  "  of  lenses,  114. 

Demandre's    method    of   controlling    tem- 
peratures of  sterilization,  12. 

Delhi  boil,  802. 

Deneke's  vibrio,  503. 

Dental  caries,  culture  medium  for,  41. 

Dermacentor  occidentalis,  802. 

Dermatitis  coccidioides,  706. 

Detection     of    micro-organisms     (see     the 
several  chapter  headings). 

Deviation  of  complement,  232. 

Dhobie  itch,  688. 
j    Diagnosis  of  glanders  with  mallein,  484. 

—  tuberculosis  with  tuberculin,  325. 
Diaphragms  for  dark-ground  illumination, 

127. 

Differential  staining  of  films  (gram -positive 
organisms),  207. 

-  —   (gram -negative      organisms), 
209. 

Differentiation  of  types  of  tubercle  bacilli 

by  cultivation,  320. 
Diphtheria  antitoxin,  265. 

—  bacillus,  245. 

—  —   non-virulent,  255. 

-  —   relationship  to  Hofmann's  bacil 
lus,  274. 

—   summary  of  diagnostic  tests,  273. 

-  contacts,  246. 

—  convalescents,  duration  of  infectivity 
246. 

-  toxin,  257. 

-  unit  of,  260. 

-  Bird,  virus  of,  842. 
Diphtheroid  organisms  in  leprosy,  351 
Diplococcus  crassus,  626. 

-  intracellularis  meningitidis,  644 
Discomyces,  655. 

—  alba,  661. 

—  astero'ides,  660,  662. 

-  bovis,  656,  661. 

—  brasiliensis,  665. 

-  caprce,  668. 

-  farcinicus,  667. 

—  fiava,  661. 

-  forsteri,  662. 

-  freeri,  664. 

-  garteni,  660,  661. 

—  hofmanni,  669. 

-  Israeli,  661. 

—  liquefaciens,  660,  661 

—  madurce,  662. 

—  minutissimus,  666. 

-  rosenbachi,  662. 

—  spitzi,  661. 

-  thibiergi,  661. 


INDEX 


875 


Discomyces,  polychrome,  669. 
Dispersive  power  of  lenses,  1 14. 
Displacement  of  air  from  culture  media,  88. 
Dissemination,  isolation  by,  77. 
Distemper,  bacillus  of,  457. 

-  (M'Gowan's),  459. 

-  filtrable  virus  in,  457,  842. 
Distortion  of  microscopical  image,  109. 
Dogs  as  experimental  animals,  157. 

-  bleeding  of,  195. 

-  handling  of,  163. 

-  inoculation     into     biliary     passages, 
177. 

-  intra-cranial  inoculation,  180. 

-  intra-spinal  inoculation,  181. 

-  in tra- venous  inoculation,  173. 

-  cesophageal  catheterization  of,  182. 

-  coccidiosis  in,  764. 

-  favus  in,  692 

-  leishmaniosis  in,  801. 

-  Nagana  in,  811. 

-  piroplasmosis  in,  791. 

—  ringworm  in,  687,  688. 

-  tuberculosis  in,  294. 
Dog-plague,  457. 
Dog-pox,  457. 

Dopter's  anti-meningococcal  serum,  649. 
Dorset's  egg  medium,  54,  316. 
Double  staining  of  sections,  219. 
Doulton's  white  porcelain  filtering  bougie, 

15. 

Dourine,  trypanosome  of,  809. 
Doyen's  coccus,  769. 
Drepanidium,  783. 

-  monilis,  784. 

—  princeps,  784. 

—  ranarum,  784. 

Dried  fruits  as  culture  media,  38. 
Drigalski's  spatula,  407. 
Dromedaries  and  El  Debab  ( Tryp.  soudan- 
ense),  813. 

—  Mbori  (Tryp.  soudanense),  813. 
Drum-stick  bacilli,  549. 

Duck  cholera,  452. 

—  pasteurellosis,  448. 
Duclaux's  filter,  25. 

—  method  for  collecting  milk,  201. 
Ducretet  and  Lejeune's  autoclave,  11. 
Ducrey's  bacillus,  513. 

Dum  dum  fever,  797  footnote. 

Dunbar's  method  for  diagnosing  cholera, 

501. 
Dunschmann's  medium  for  isolation  of  the 

typhoid  bacillus,  410. 

—  method  for  isolation  of  the  typhoid 
bacillus  from  blood,  392. 

-  for     preparation     of     toxin     of 
quarter  ill,  558. 

-  serum  for  quarter  ill,  559. 
Duval's  media  for  the  leprosy  bacillus,  352. 
Dyes,  aniline  (see  Stains). 

Dysentery,  amoebae  in,  746. 

-  trichomonas  in,  825. 

-  serum  diagnosis  of,  364. 

-  serum  therapy  of,  361. 

-  vaccination  against,  362. 

—  bacillus,  356. 


|   Dysentery,  amoebae  in,  El  Tor,  365. 

-  classification  of,  357,  360. 
Dysgonic  tubercle  bacilli,  314. 

East  African  relapsing  fever,  712. 
East  Coast  fever  of  cattle,  793. 
Echincoccus  infection  in  cattle,  Tuberculin 

in,  326. 

Eczema  marginatum,  688. 
Egg  as  a  culture  medium,  53,  316,  317. 
Egg-albumin  (powdered).  639. 
Ehrlich's  aniline- violet,  139. 

—  method   of  standardizing   antitoxin, 
268. 

—  stain  for  tubercle  bacilli,  308,  311. 

—  theory  of  bacteriolysis,  229. 
Ehrlich-Biondi  stain,  768  footnote. 
Eimeria,  764. 

—  hominis,  764. 
El  Debab,  813. 

El  Tabardillo,  847. 

El  Tor  bacillus,  365. 

Eleidine,  766. 

Elephants  and  Surra,  814. 

Ellermann    and    Erlandsen's    method    for 

examination   of  sputum   for   tubercle 

bacilli,  339. 

Elmassian's  bacillus,  510. 
Eisner's  potato  gelatin,  41. 

—  method    for    isolating    the    typhoid 
bacillus,  85,  403. 

Embedding  methods,  212. 
Endo's  agar,  408. 
Endodermophyton  concentricum,  687. 

—  indicum,  688. 
Endolysins,  223. 
Endomyces,  701  footnote. 

—  albicans,  702. 
Endospore,  145. 
Endotoxin,  cholera,  495. 

—  dysentery,  361. 

—  plague,  467. 

—  typhoid  (Besredka's),  379. 
Entamoeba  coli,  747. 

—  histolytica,  747. 
Enteric  fever,  366. 

-  serum  diagnosis  of,  384. 
and  flies,  367. 

Enterococcus,  627. 

Enumeration  of  organisms  in  water,  853. 

Eosin,  alcoholic  solution,  209. 

-  aqueous  solution,  207,  218. 
Epidemic  cerebro- spinal  meningitis,  644. 

—  —   (pneumococcal),  581. 
Epidermophyton  cruris,  688. 

-  gallince,  692. 

—  inguinale,  688. 
Epilation  of  hair,  170. 
Epizootic  lymphangitis,  706. 
Epizootics  (spontaneous)  among  laboratory 

animals,  159. 

-  due  to  anthrax,  518. 

—  —   —    B.  typhi  murium,  444. 

-  filter  passing  organisms,  160, 
835  et  seq. 

-  Gaertner's  bacillus,  442. 

—  —  —   Microsporidia,  752. 


876 


INDEX 


Epizootics  (spontaneous)  due  to  Pasteurella 
group,  446  et  seq. 

—   Plague  bacillus,  460. 

—  (experimental)  among  ground  squir- 
rels, 448. 

Eppendorf's  whooping  cough  bacillus,  570. 
Eppinger's  streptothrix,  662. 
Epstein's  stain  for  diphtheria,  253. 
Equidse  and  Mai  de  Caderas,  814. 
Equine  herpes,  687,  689. 

-  pasteurella,  456. 

-  piroplasmosis,  792. 

—  tuberculosis,  296. 
Ericolin,  352. 
Erlenmeyer  flask,  29. 

van  Ermengem's  stain  for  flagella,  149. 
Ernst-Neisser  stain  for  diphtheria,  252. 
Erysipelas,  592. 
Erysipele,  283  footnote. 
Erysipelococcus,  593. 
Erythrasma,  666. 
Esmarch's  tubes,  103. 

—  roll  tubes,  81. 
Espundia,  802. 

Ether  as  an  antiseptic,  27. 

—  method  for  embedding,  215. 

—  temperature  regulators,  59. 
Eugonic  tubercle  bacillus,  314. 
European  relapsing  fever,  712. 
Ewes,  gangrenous  mammitis  of,  615. 
Examination   (macroscopical)   of  cultures, 

73. 

-  of  stained  preparations,  135. 

-  of  unstained  preparations,  131. 

—  (bacteriological)  of  fluids  and  tissues, 
203. 

Excreta  (see  Stools). 
Exudates,  inoculation  of,  169. 
Exoascidce,  701  footnote. 
Experimental    animals,    spontaneous    dis- 
eases of,  159. 

-  inoculation  (see  the  several  chapter 
headings). 

Eye,  inoculation  into  anterior  chamber  of, 

178. 
Eye  pieces,  120. 

—  compensating,  115. 

—  Huygenian,  116. 

—  Ramsden,  122. 

Eyre's  method  for  neutralization  of  culture 
media,  31. 

Facultative  anaerobes,  87. 

Faeces  (see  Stools). 

False  membrane,  collection  of,  197,  270. 

-  produced  by  toxin,  261. 

-  spores,  526. 
Farcy,  480. 

-  buds,  480. 

—  Japan,  707. 

Fasoti's  stain  (Negri  bodies),  841. 
Favus,  690. 

—  in  the  lower  animals,  692. 
Fehleisen's  Streptococcus  erysipelatos,  593, 

596. 

Femoral  artery,  inoculation  into,  173. 
Fernbach  flask,  253. 


Ferran's  cholera  vaccine,  496. 
Ferret  septicaemia,  bacillus  of,  453. 
Ferrets  and  plague,  461. 
Ficker's  methods  for  the  isolation  of  the 
typhoid  bacillus,  406,  409. 

-  typhus  diagnosticum,  388. 
Film  preparations,  203. 

—   staining  of,  204. 

-  differential  staining  of,  207. 

-   for  gram -negative  organ- 
isms, 210. 
Filters,  14. 

-  for  small  quantities  of  fluids,  24. 

—  collodion  sacs  as,  175. 
Filtrable  viruses,  835. 

—  —   and     dark-ground     illumination, 
124. 

-  as  causes  of  epizootics,  160,  439, 
835  et  seq. 

Filtration  by  aspiration,  19. 

-  compression,  18, 

—  of  agar,  Fischer's  method,  44. 

-  —  Karlinski's  method,  44. 

—  carbohydrate  media,  35. 

—  culture  media,  18. 

—  serum,  47. 

-  (Miquel's  apparatus),  48. 

-  small  quantities  of  fluid,  24. 

-  spirochaetes,  713. 

—  Treponema  pallidum,  737. 

—  water,  15. 
Finkler-Prior  vibrio,  502. 

Fischer's  method  for  nitration  of  agar,  44. 

—  gelatin,  41 

Fish,  Microsporidia  in,  754. 

—  Myxosporidia  in,  756. 

—  Trypanosomes  in,  824. 

—  Tuberculosis  in,  296. 
Fixateur,  229. 
Fixation  of  films,  205. 

—  complement,  232. 
Fixative,  albumin  (Mayer's),  215. 

-  Borrel's  for  sporozoa  in  sections,  761. 

-  Pianese's  for  coccidia,  761. 

-  Schaudinn's,  749. 
Fixing  reagents  for  tissues,  188. 
Flagellata,  803. 

Flagellated  malarial  parasite,  775. 
Flagellum  staining,  148. 
Flaschen  bacillen  of  Unna,  693. 
Flatness  of  image,  115. 
Fleig's  test  for  indol,  374. 
Flemming's   solution   as   hardening   agent, 
189. 

—  perchloride  as  hardening  agent,  190. 
Flexner's  dysentery  bacillus,  357. 

-  antimeningococcal  serum,  648. 
Fleas  and  Dourine,  809. 

—  Plague,  461. 

-  Trypanosomiasis,  805. 

Flies  and  Anterior  poliomyelitis,  846. 

—  Enteric  fever,  367. 

-  equine  Piroplasmosis,  792. 

-  Galziekte,  816. 

—  Kala  azar,  800. 

-  Oriental  sore,  802. 

-  Plague,  462. 


INDEX 


877 


Flies  and  Surra,  814. 

Fluids,  bacteriological  examination  of,  203. 

Fluorescein,  aqueous  solution,  218. 

"  Fly  disease,"  813. 

Foa's  differential  stain  for  sections,  220. 

Focal  length  of  lenses  and  magnification, 

120. 
Food  poisoning,  438. 

-  (Aertrycke  bacillus),  438. 
(B.  botulinus),  549. 

-  (Gaertner  bacillus),  442. 

-  (Paratyphoid  B.  bacillus),  432. 
Food  stuffs  and  plague,  462. 

Foot  and  mouth  disease,  virus  of,  838. 
Forceps,  Cornet's,  131. 

-  Debrand's,  131. 

—  Listen's,  187. 
Formalin  as  an  antiseptic,  26. 

-  hardening  agent,  189. 

Fowls,    epizootic    among    (Pasteurella   gal- 
lince),  447. 

—  —   (Vib.  metchnikowi),  503. 

—  favus  in,  692. 

—  ringworm  in,  687. 

—  spirochsetosis  in,  718. 

—  and  plague,  461. 
Fowl  cholera,  bacillus  of,  447. 

-  plague,  447. 

-  septicaemia,  447. 

-  typhoid,  447. 
Fractional  cultivation,  84. 
Frankel's  stain  (Tubercle  bacilli),  309. 

—  method  for  isolating  anaerobes,  103. 
Frambcesia,  736. 

Frankland's    method    for    examination    of 

air,  865. 

Freezing  methods  for  cutting  sections,  212. 
Friedberger's   B.   hcemoglobinophilus  canis, 

510. 
Friedberger    and    Moreschi's    antityphoid 

vaccination,  381. 
Friedlander's  bacillus,  415. 

—  stain  (Pneumococcus),  584. 
Frogs  as  experimental  animals,  157. 

-  feeding  of  (Ledoux-Lebard),  158. 

-  keeping  of,  158. 

-  inoculation  of,  171. 

-  Trypanosomes  in,  824. 
Fruits  as  culture  media,  38. 
Fuchsin  ink,  150. 
Furnace,  muffle,  17. 
Furunculosis,  617. 

Fuscus  crispus,  746. 

Gabbe's  stain  (Tubercle  bacilli),  308. 

Gaertner' s  bacillus,  442. 

Gall  sickness,  816. 

Galziekte,  816. 

Gamaleia's  cholera  toxins,  495. 

Gangrene,  anaerobic  organisms  in,  569. 

—  gaseous,  561. 

Garlic,  essence  of,  as  an  antiseptic,  27. 

Garotilho,  517. 

Garros'  filtering  bougie,  15. 

Gases,  sterilization  of,  89. 

Gasser's  culture  medium,  57. 

Gastro-enteritis  of  dogs,  457. 


Gathgens'  method  for  the  isolation  of  the 

typhoid  bacillus,  409. 
Geese,   epizootics    among   (Past,    gallince), 

448. 

—  osteo-myelitis  in,  618,  619. 

—  spirochsetosis  in,  717. 

—  and  Plague,  461. 
Gelatin  culture  media,  39. 

-  types  of  growth  in,  74. 

—  —  liquefaction,  74. 

—  -agar,  43. 

—  litmus,  57. 

—  glucose-litmus,  57. 

—  lactose-litmus,  57. 

—  mannite-litmus,  57. 

-  raisin,  41. 

Gemelli's  stain  for  flagella,  153. 
German  plague  Commission  vaccine,  469. 
Giblet  broth,  32. 

Giemsa's  solution,  727  footnote,  774. 
Gioelli's  placenta  medium  (Tubercle  bacil- 
lus), 318. 
Glanders  bacillus,  480. 

-  latent,  485. 

Glass  for  lenses,  114,  116. 

Glass  needles  for  sowing  cultures,  70. 

Glossina  morsitans  and  Nagana,  811. 

-  and  Sleeping  Sickness,  820,  821. 

-  palpalis  and  Nagana,  811. 

—   and  Sleeping  Sickness,  820. 
Glosso-anthrax,  518. 
Glucose  for  absorbing  oxygen,  89. 

—  in  cultivation  of  anaerobes,  99. 

—  -glycerin -agar,  44. 
glycerin-gelatin,  664. 

Glugea  bombycis,  752. 
Glycerin-agar,  44,  693. 
broth,  35. 

—  -fish-broth  (Tubercle  bacillus),  319. 

—  -potato,  55. 

-  for  tubercle  bacilli,  320. 

—  -serum,  53. 

Goats  as  experimental  animals,  156. 

—  Mediterranean  fever  in,  475. 

-  Pasteurellosis  in,  456. 

-  Sarcosporidiosis  in,  758. 

-  Tuberculosis  in,  295. 
Golaz's  wax,  53. 
Gonococcus,  634. 

-  and  Meningococcus,   similarities  be- 
tween, 645. 

-  blood-broth  for,  34. 
Gonotoxin,  640. 
Gonospora  longissima,  794. 

Gordon's  medium  (differentiation  strepto- 
cocci), 35. 

—  metabolic  tests,  601. 
Gorsline's  collodion  sacs,  176. 
Gosio's  plague  vaccine,  469. 
"  Gout "  in  swine,  283,  286. 
Gram's  solution,  143. 

—  stain,  142. 
for  films,  207. 

—  —   for   typhoid    bacilli    in    sections, 
217. 

"  Grapes,"  294. 
Grasbazillus,  345. 


878 


INDEX 


Grassi's  solution,  748. 

Grassberger     and     Schattenfroh's     serum 

(quarter  ill),  560. 
Gregarina  blattarum,  796. 
Gregarinida,  794. 
Griffith's    (A.    S.)    egg    medium    (Tubercle 

bacillus),  317. 

-  glycerin-potato    (Tubercle    bacil- 
lus), 321. 

-  —  method  isolation  tubercle  bacilli 
from  sputum,  341. 

—  (A.    S.    and    F.),    immunization    of 
cattle,  331. 

Grijns'  agar  for  A.  fumigatus,  697. 
Grimbert's  medium  for  fermentation  reac- 
tions, 417. 

-  isolation     of     typhoid     bacillus, 
404. 

Grimbert  and  Legros'  medium,  373. 
Ground  squirrels  as  experimental  animals, 
157. 

-  epizootics  among  (B.  typhi  murium), 
444. 

—  extermination  of,  448. 

—  natural  plague  in,  461. 

—  Trypanosom.es  in,  808. 
Griinbaum-Widal  reaction,  384. 
Group-agglutinins  (see  Co-agglutinins). 
Gueniot's  placenta  medium,  54. 

Guinea     fowl,     epizootics     among     (Past. 

gallince),  447. 
Guinea-pigs  as  experimental  animals,  156. 

-  collection  of  blood  from,  194. 

—  epizootics   among   (Gaertner's   bacil- 
lus), 442. 

—   (filter-passing  organisms),  439. 

-  (infectious  pneumonia),  457. 

-  (spontaneous  plague),  460. 

-  handling  of,  162. 

-  inoculation  into  biliary  passages,  177. 

-  intra-cranial  inoculation,  180. 

—  oesophageal  catheterization,  181. 

—  intra-spinal  inoculation,  181. 

-  in tra- venous  inoculation,  172. 

—  Trypanosomes  in,  808. 
Gummata,  multiple  disseminated,  672. 
Gunther's  stain  (Spirochaetes),  715. 
Gymnoascidce,  679. 

Hcemacytozoa,  770. 
Hcemamceba,  770. 

—  danilewskyi,  782. 

—  kochi,  781. 

—  mqjoris,  782. 

—  malar  ice,  770. 

-  —  var.  parva,  780. 
tertiana,  780. 

—  —  —  quartana,  780. 

-  melaniphera,  781. 

—  relicta,  782. 

—  ziemanni,  782. 

Hcetnaphysalis  leachi  and  Piroplasma  canis, 
791. 

—  punctata  and  Piroplasma  bigeminum, 
787. 

Hsematein  solution,  218. 
Hcematococcus,  786  footnote. 


Hcematopinus  spinulosus  and  Trypanosoma 

lewisi,  805. 

Hsematoxylin,  Bcehmer's,  218. 
Haematozoa,  770. 

—  of  birds,  781. 

—  of  malaria,  778. 
Haematozoon  of  bats,  781. 

—  monkeys,  781. 
Haemoglobin  for  blood-broth,  34. 

—  blood-agar,  53. 
Hcemogregarina,  783. 

—  lacertarum,  785. 

—  ranarum,  784. 

—  stepanowi,  783. 
Haemolysin,  streptococcal,  603. 
Hsemolysins,  230. 
Hsemolyso-diagnosis,  238. 
Hsemolytic  couple,  232. 
Hsemolytic  serum,  titration  of,  234. 
Hsemorrhagic   septicaemias,   bacilli   of  the, 

421,  446. 
Haffkine's  cholera  vaccine,  497. 

—  plague  vaccine,  468. 
Hagemann's  agar,  408. 

Hair,     collection     of,     for     bacteriological 
examination,  191. 

—  epilation  of,  170. 
Halteridium,  770. 

—  danilewskyi,  782. 

Handling  of  experimental  animals,  160. 
Hanging-drop  preparations,  132. 
Hansen's  bacillus,  348. 
Haplosporidia,  759. 

Hardening  reagents  for  tissues"  (see  Fixa- 
tives), 188. 

Haricot  decoction  medium,  37. 
Hay  infusion  medium,  37. 
Heart-water,  839. 
Heat  as  an  auxiliary  to  staining,  137. 

-  fixation  of  films  by,  205. 

-  resistance  of  bacteria  to,  7. 

—  —  spores  to,  8. 

-  sterilization  by  discontinuous,  8,  12. 
dry,  4. 

—  —   moist,  7. 

Hecht  and  Wilenko's  stain  (T.  pallidum), 

728. 

Hectic  fever,  297. 
Heiman's  agar,.  639. 
Heinemann's  jelly,  56. 
Helcosoma  tropicum,  802. 
Hereditary  infection  in  Noserna  apis,  754. 

—  — *  —   bombycis,  753. 

—  —   lice  (spirochaetosis),  712. 

—  —   ticks  (spirochaetosis),  712. 

—   (piroplasmosis),  787,  791. 
Herman's    stain    (Tubercle    bacilli),    309, 

311. 
Herpetomonas,  804. 

-  lewisi,  805  footnote. 

—  muscce  domesticce,  805  footnote 
Herxheimer  s  stain  (T.  pallidum),  729. 
Herxheimer  and   Huber's  stain  (T.  palli- 
dum), 729. 

Hesse's   albumose   agar   (tubercle    bacilli), 

0 1  / . 

-  peptone  agar,  410. 


INDEX 


879 


Hesse's  method  (Examination  of  air),  863. 

(Cultivation    of    tubercle    bacilli 

from  sputum),  340. 

—  tube,  863. 

Heterologous     agglutinins     (see    Co-agglu- 

tinins). 

Hey  den's  albumose,  340. 
Hippoboscidce    and    Trypanosoma    theileri, 

816. 
Hiss'  serum -water  medium,  585. 

-  stain  for  capsules,  148. 

—  classification    of    dysentery    bacilli, 
360. 

-  Y  bacillus,  360,  363. 

Histological  examination,   removal  of  tis- 
sues for,  188. 

-  preparations  (see  Sections),  211. 

-  (freezing  methods),  212. 

-  (paraffin  embedding),  212. 

—  —   simple  staining  of,  216. 

—  —   differential  staining  of,  216. 
Hofmann's  bacillus,  273. 

Hoffmann  and  Halle's  stain  (T.  pallidum), 

729. 
Hog  cholera,  439. 

-  —  virus  of.  843. 

-  and  swine  plague,  454. 

-  and  swine  erysipelas,  288. 
Hollow  ground  slides,  132. 

—   cultivation  in,  134. 
Homologous  agglutinins  (see  Agglutinins). 
Horse  plasma  (Jousset's),  342. 

-  serum  and  anaphylaxis,  224. 

-  sickness,  virus  of,  838. 

-  syphilis,  809. 

—  typhoid,  456. 

Horses  as  experimental  animals,  156. 

—  collection  of  blood  from,  48,  193. 

—  handling  of,  164. 

—  intra- venous  inoculation  of,  173. 

-  anthrax  in,  518. 

-  piroplasmosis  in,  792. 

—  ringworm  in,  687. 

-  spirochsetosis  in,  719. 

-  tetanus  in,  536. 

—  trypanosomiasis    in,    809,    811,    813, 
814. 

—  tuberculosis  in,  296. 
Hospital  gangrene,  574. 
Hot  water  funnel,  40. 
Hot  air  sterilizers,  5. 

Houses  for  experimental  animals,  157. 
Houston's  method  of  water  examination, 

858. 

Huber's  haemoglobin-agar,  507. 
Hueppe  and  Scholl's  cholera  toxin,  495. 
Human  serum  therapy  (see  Serum  therapy). 

—  tubercle  bacillus,  289. 

—  tuberculosis,  292. 

—  vaccine       therapy       (see       Vaccine 
therapy). 

Huygenian  eyepiece,  116. 

—  —   not   suitable   for   measuring   ob- 
jects, 122. 

Hyalomma    cegyptium    and    Bovine    farcv, 

667. 
Hvdrochloric  acid  solution,  210. 


Hydrogen,  generation  of,  88. 

-  (in  cultivation  of  anaerobes),  88, 
89. 

Hydrophobia,  virus  of,  841. 
Hyperimmunization,  222.- 
Hypomycetes,  655. 

Iceland  moss  as  substitute  for  agar,  44. 
Ichthic  tubercle  bacillus,  292,  296. 
Identification  of  antibodies  by  complement - 
fixation,  238. 

-  organisms    (see   the   several   chapter 
headings). 

-  by     complement-fixation,      237, 
437. 

Idiopathic  tetanus,  536. 

I.E.,  268. 

Ilkewitsch's    method    for    examination    of 

sputum  (tubercle  bacillus),  339. 
Image,  brightness  of,  112. 

-  flatness  of,  115. 

-  sharpness  of,  114. 
Immune  body,  228. 
Immunity,  221. 

—  acquired,  221. 

—  crossed,  in  spirochaete  infections,  717. 

—  mechanism  of,  222. 

—  natural,  221. 

—  passive,  222. 

Immunization  (see  Vaccination). 
Inactivated  serum,  234. 
Incinerator,  17. 

Incubators,  58. 

—  opsonic,  240. 

—  vacuum,  104. 
Incubator  rooms,  59. 

India-rubber  apparatus,  sterilization  of,  9. 

—  caps,  29. 
Indian  kala  azar,  797. 

compared    with    Mediterran- 
ean, 800. 

—  relapsing  fever,  712. 
Indiella,  665  footnote. 

—  mansoni,  665. 

—  reynieri,  665. 

—  somaliensis,  665. 
Indigo  white,  88,  92. 

—  a  test  for  oxygen,  92. 
Indol,  medium  for  production  of,  374. 

—  tests  for,  374. 
Infantile  diarrhoea,  356. 

—  kala  azar,  800. 

-  paralysis,  844. 

—  splenic  anaemia,  800. 
Infection  by  feeding,  181. 

—  ingestion,  181. 

—  inhalation,  180. 

*'  Infectious  epithelioses,"  840. 
Infectious  pneumonia  of  dogs,  457. 

goats,  456. 

Influenza  bacillus,  504. 

—  of  horses,  456. 
Infusoria,  829. 
Infusorial  earth  filters,  15. 
Ingestion,  infection  by,  181. 
Inhalation,  infection  by,  180. 

—  theory  of  tuberculosis,  292. 


880 


INDEX 


Inoculated    animals,     observations    upon, 

182. 
Inoculation  of  animals,  156. 

—  general  rules  for,  170. 

—  preparation  of  material  for,  169. 

—  apparatus  for,  of  large  quantities  of 
fluid,  168. 

-  instruments  for,  165. 

—  of  mucous  surfaces,  171. 

—  needles,  168. 

-  Pasteur  pipettes  for,  166. 

—  subcutaneous,  171. 

-  syringes,  166. 

—  into  intestines,  182. 

-  lymph  spaces,  171. 
Instruments  used  for  inoculation,  165. 

-  post  mortem  examinations,  184. 

—  —  sowing  cultures,  67. 
Internal  organs  as  culture  media,  54. 
Intestinal  amoebae,  746. 

—  anthrax,  517. 
Intra-arterial    inoculation,  173. 
Intra-cerebral  181. 
Intra-cranial  180. 
Intra-dermal                           171. 
Intra-muscular        —  172. 
Intra-peritoneal  174. 
Intra-pleural  179. 
Intra-pulmonary  179. 
Intra-spinal  181. 
Intra-tracheal         —           179. 
Intra- venous                           172. 
Intra-dermo  reaction  (Tuberculosis),  327. 
Inoscopy,  342. 

Invisible  micro-organisms,  835. 
Iodine  solution  (Gram's),  143. 

-  (Merieux's),  209. 
(Nicolle's),  209. 

Irregular  malarial  fever,  780. 
Isle  of  Wight  bee  disease,  752. 
Isolation  of  aerobic  micro-organisms,  76. 

—  anaerobic  micro-organisms,  101. 
Issaeff  s  pneumococcus  toxin,  587. 
Ixodes  ricinus  and  Piroplasma  bigeminum, 

787. 
Ixodioplasma,  786  footnote. 

Jackson  and  Melia's  method  of  isolating  the 

typhoid  bacillus,  410. 
Jaeger- Heubner  coccus,  626. 
Jatta  and  Maggiora's  plague  vaccine,  469. 
Jamamoto's  stain  (Leprosy  bacillus),  350. 
Japan  farcy,  707. 
Jena  flasks,  29. 
Jenner's  stain,  773. 
Jockmann's    method    of    cultivating    the 

tubercle  bacillus  from  sputum,  340. 
Jousset's  methods  of  detecting  the  tubercle 

bacillus,  340,  342,  344. 
Jugular  vein,  inoculation  into,  173. 
Jurewitch's  potato  medium  for  the  tubercle 

bacillus,  320. 

Kala  azar,  Indian,  797. 

—  infantile,  800. 

—  Mediterranean,  800. 
Kangaroo,  Sarcosporidia  in  the,  758. 


Karlinski's  filter  for  agar,  44. 

Kayser's  method  for  isolating  the  typhoid 

bacillus  from  blood,  392. 
Keeping  of  animals,  157. 
Kitasato's  dish,  101. 

—  filter,  25. 

-  method  for  collecting  sputum,  192. 

—  —   cultivating  anaerobes,  99. 

-  tubercle  bacilli  from  sputum, 
340. 

—  isolating  anaerobes,  101. 

-  vaccinating    against    quarter   ill, 
557. 

Kidney,  inoculation  into,  178. 

Kitt's  quarter  ill  serum,  559. 

Klausner's    serum    diagnosis    of    Syphilis, 

741. 

Klebs-Lceffler  bacillus,  245. 
Klingmueller's  phenomenon,  327. 
Klemperer's  pneumococcus  toxin,  586. 
Klossia  helicina,  765. 
Koch's    bacillus     of     mouse     septicaemia, 

288. 

-  drying  stage,  141. 

-  hollow-ground  slide,  132. 

-  method  for  collecting  serum,  46. 

-  examination  of  air,  863. 

—  isolation  of  aerobes,  77,  79,  85. 

—  old  tuberculin,  324. 

-  peptone  solution,  33. 

—  serum  coagulator,  51.   . 

—  stain  (Tubercle  bacilli),  310. 

—  steamer,  8. 

—  vibrio,  488. 

Kolle's  cholera  vaccine,  497. 

Kolle  and  Otto's  plague  vaccine,  469. 

Krai's  agar  for  gonococcus,  639. 

Krauss  and  Doerr's  dysentery  serum,  362. 

Krawkoff's  cholera  toxin,  495. 

Kiihne's  alkaline  blue.  139. 

-  carbol-methylene-blue,  138. 

—  differential  stain  for  films,  210. 

-  sections,  219. 

-  simple  stain  for  sections,  216. 

-  stain  (Tubercle  bacilli),  310. 
Kurth's  Streptococcus  conglomeratus,  596. 

La  bacteridie  ovoide,  446. 
'"  Laboratory  animals,"  156. 

—  spontaneous  diseases  of,  159. 
La  clavelee,  839. 

Lacomme's  tube  (cultivation  of  anaerobes), 
93. 

Lafforgue's   method   (isolation  of   typhoid 
bacillus  from  blood),  391. 

Lamblia  intestinalis,  827. 

Lankesteria  ascidice,  796. 
!   La  poliomyelite  epidemique,  844. 

Largine  for  staining  Treponema  pallidum, 
728. 

Latapie's    apparatus    for    bleeding    large 
animals,  50. 

—   small  animals,  196. 

Laveran's  method  (bacteriological  examina- 
tion of  air),  867. 

—  blood  stain,  210. 

-  stain  (Malaria),  772,  773. 


INDEX 


881 


Lavcran     and     Pettit's     culture     medium 

(Leishmania  donovani),  799. 
Laverania  malarice,  770,  780. 
Le   Dantec's  method  of  examining  blood 

(Malaria),  772. 

Leclainche's  malignant  oedema  serum,  568. 
Leclainche  and  Vallee's  quarter  ill  serum, 

559. 

—  —   malignant  oedema  vaccines,  565, 
568. 

—  —   quarter  ill  vaccines,  557-8. 
Ledoux-Lebard's  method  of  feeding  frogs, 

158. 

"  Leeches,"  674. 
Leeches  as  intermediate  hosts  of  haemogre- 

garines,  784. 

-   H.  ranarum,  785. 
Legerella,  766. 
Legros'  method  for  cultivating  anaerobes, 

97. 

Leipschutz's  agar  (Gonococcus),  639. 
Leishman's  stain,  773. 
Leishman-Donovan  body,  797. 
Leishmania  donovani,  797. 

—  furunculosa,  802. 

—  infantum,  800. 

—  tropica,  802. 

-  —   var.  americana,  802. 
Leishmaniosis  in  cats,  802. 

-  dogs,  801. 
Lemco,  34. 

Lemiere  and  Becue's  stain  (Actinomycosis), 

658. 
Lenses,  apochromatic,  115. 

—  correction  of,  115. 

—  definition  of,  114. 

-  homogeneous  immersion,  119. 

—  penetrating  power  of,  114. 

-  resolving  power  of,  114. 

-  methods  of  testing  definition  of,  114. 
Lepidophyton  concentricum,  700. 
Lepidoptera,  microsporidia  in,  754. 
Lepine  and  Lyonnet's  typhoid  toxin,  376. 
Lepra  cells,  354. 

Leprosy  bacillus,  306,  348. 

—  and    tuberculosis,    simultaneous    in- 
fection with,  355. 

Leptotheca  agilis,  756. 

Lesage's  method  for  cultivation  of  amoebae, 
750. 

Letulle's  stain  (Tubercle  bacilli),  311. 

Leuch's  method  for  isolation  of  the 
typhoid  bacillus,  409. 

Leuckart's  moulds  for  paraffin  embedding, 
214. 

Leucocidine,  623. 

Leucocytozoon  canis,  783. 

Leucotoxin  in  treatment  of  leprosy,  354. 

Leukins,  223. 

Levaditi's  pipettes,  234. 

Levaditi's  stain  (T.  pallidum),  731. 

Levaditi  and  Manouelian's  stain  (T.  palli- 
dum), 731. 

Liborius'  agar  for  anaerobic  cultivation, 
99. 

Liborius  and  Veillon's  method  of  isolating 
anaerobes,  103. 


Lice  and  spirochaetosis,  711. 

—  trypanosomiasis,  805. 

—  typhus,  848. 

Lichen  crispus,  an  agar  substitute,  44. 
Lichtheimia  corymbifera,  677. 

—  racemosa,  677. 
Liebig's  broth,  34. 

—  gelatin,  41. 

Life  history  of  Coccidium  cuniculi,  762. 

-  Gregarinida,  794. 

—  —   Leishmania  donovani,  799. 

-  —   malarial  parasite,  776. 

-  Nosema  apis,  754. 

-  bombycis,  752. 

-  Piroplasma  bigeminum   788. 
—   canis,  792. 

—  —  Sarcosporidia,  757. 

—  —    Trypanosoma  cruzi,  822. 
Lignieres'    polyvalent   vaccine    (pasteurel- 

loses),  459. 
von  Lingelsheim's  Streptococcus  brevis,  596. 

-  longus,  596. 

Liquefaction  of  gelatin,  types  of,  74. 
Liquid  culture  media,  30. 

Lister's  method  of  isolating  organisms,  76. 

Liston's  forceps,  187. 

Lithia  solution  (Kiihne's),  210. 

Litmus  as  an  indicator,  31,  56. 

-  solution,  preparation  of,  56. 
Lceffler's  aniline-fuchsin,  483. 

—  methylene  blue,  139. 

-  serum,  52. 

-  for  diphtheria,  271. 

—  bile    medium    for    isolation    of    the 
typhoid  bacillus,  410. 

—  malachite -green  for  isolation  of  the 
typhoid  bacillus,  410. 

-  stain  for  flagella,  149. 

-  sections,  216. 
Lophophyton  gallince,  692. 

Lorrain  Smith's  medium  (diphtheria  bacil- 
lus), 271. 

Lowenberg's  bacillus,  419. 
Lubenau's  egg  medium,  54. 

-  caffeine-agar  (typhoid  bacillus),  409. 
Lugol's  solution  as  a  mordant,  143. 
Lumbar  puncture,  199. 

Lumiere's  bleeding  apparatus,  196. 

-  (A.    and    L.)    tissue    culture    media 
(tubercle  bacillus),  318. 

Lung,  inoculation  into,  179. 

Lupus,    characteristics   of   bacilli   in,    290,. 
293. 

Lustigarten's  stain  (Tubercle  bacilli),  310, 
311. 
"  Syphilis  bacillus,"  346. 

Lustig  and  Galeotti's  plague  vaccine,  470. 

Lycoperdon  spores,  180. 

Lycopodium  powder,  180. 

Lymph  spaces,  inoculation  into,  171. 

Lymphatic    glands,    removal   of,    for   bac- 
teriological examination,  198. 

Lysol  as  an  antiseptic,  26. 

/j.  as  a  unit  of  measurement,  122. 
MacConkey's  media  (typhoid-colon  group)* 
85,  412. 


882 


INDEX 


Macfadven  and  Rowland's  typhoid  toxin, 

379. 

M'Gowan's  bacillus  of  distemper,  459. 
Madura  foot,  662. 

-  —  parasites  of,  665. 
Madurella  (genus),  665  footnote. 
Magnification,  limits  of  effective,  113. 

—  measurement  of,  122. 

—  produced  by  a  microscope,  107. 
.Mai  de  brou,  787.' 

-  de  Caderas,  814. 

-  du  coit,  809. 

—  de  la  Zousfana,  813. 
Malachite  green  media,  375,  409. 
Maladie  de  Heine-Medin,  844. 
Malarial  fevers,  types  of,  780. 
Malassez's  syringe,  167. 

-  warm  stage  for  microscope,  135. 
Malassezia  furfur,  669. 

—  macfadyeni,  670. 

—  mansoni,  670. 

—  tropica,  670. 
Malignant  jaundice  of  dogs,  791. 

-  oadema,  vaccine  for,  565. 

—  pustule,  517. 
Malignes  Odem,  561. 
Mallein,  484. 

-  in  diagnosis  of  glanders,  484. 

-  and  tuberculin,  similarity  of  effects 
of,  325. 

Mallory  and  Wright's  stain  (Amoebae),  749. 

Malm's  agar,  44. 

Malt  extract  medium,  37. 

Malta  fever,  475. 

Malta  vibrio,  492. 

Mammalia,    post   mortem    examination    of, 

187. 

Mammary  tuberculosis  in  cows,  294. 
Mammitis  of  cows,  streptococcus  of,  613. 

—  ewes,  micrococcus  of,  615. 
Manchuria,  epidemic  plague  in,  462. 

-  treatment  of  dysentery  in,  362. 
Mannite -fermenting  dysentery  bacilli,  357. 
Manure  bacillus,  345. 

Maragliano's  antituberculous  serum,  334. 

-  tuberculin,  329. 
Marchoux's  antianthrax  serum,  531. 

-  medium  for  virus  of  bird  plague,  839. 
Marino's   method   for  isolating  anaerobes, 

102. 

-  stain  (T.  pallidum),  727. 
Marmier's  anthrax  toxin,  529. 
Marmorek's  phenomenon,  598. 

-  antistreptococcus  serum,  606. 
Marmots  and  plague,  461,  462. 
Martin's  diphtheria  toxin,  257. 

-  glycerin-fish-broth    (Tubercle    bacil- 
lus), 319. 

-  (E.)  filtering  apparatus,  25. 

-  (L.)  filtering  apparatus,  23. 

-  peptone  broth,  33. 
—   solution,  32. 

Martini  and  Lentz's  antidysenterv  serum, 

362. 

Massaouah  vibrio,  490,  491,  492. 
Massol's  diphtheria  toxin,  259. 
Mayer's  hardening  solution,  189. 


Mayer's  albumin  fixative,  215. 

Maze's  medium  for  bacteria  of  plants,  38. 

Mbori,  813. 

Measles,  swine,  283. 

Measurement  of  microscopical  objects,  121. 

Meat  as  a  culture  medium,  54. 

—  extract,  32. 
Mechanism  of  agglutination,  226. 

—  bacteriolysis,  228. 

—  haemolysis,  231. 

—  immunity,  222. 
Media  (see  Culture  media),  28. 
Mediterranean  fever,  475. 

—  kala  azar,  800. 
Megastoma  entericum,  827 
Melanin,  774. 
Melanoid  mycetoma,  665. 
Meningitis,  meningococcal,  644. 
Meningococcus,  644. 

-  and  gonococcus  compared,  645. 

—  blood-broth  medium  for,  34. 
Meningococcal  meningitis,  644. 
Mentagra,  686. 

Mercury  oxycyanide  as  antiseptic,  26. 

-  perchloride  as  antiseptic,  26. 

-  as  hardening  reagent,  189. 

-  pump,  90. 

Merieux's  differential  stain  for  films,  209 

—  iodine  solution,  209. 
Metabolic  tests,  Gordon's,  601. 

Metal   instruments,    sterilization   of,   4,    9, 

26. 
Metchnikoffs    method    for    isolating    the 

cholera  vibrio,  85,  501. 

-  exterminating    ground    squirrels, 
448. 

-  peptone -gelatin      medium      (cholera 
vibrio),  33. 

-  vibrio,  503. 

Metchnikoff,  Roux  and  Salimbeni's  cholera 
serum,  499. 

-  collodion  sacs,  174. 
Methaemoglobineemia,  spontaneous,  in  rats, 

442. 

Methods   of  abstracting  air  from   culture 
media,  87. 

-  controlling  the  temperature  of  steri- 
lization, 12. 

-  examining  air,  862. 

-  tissues,  etc.,  for  micro-organisms 
(see  the  several  chapter  headings). 

-   —   water,  853. 

-  isolating  aerobic  micro-organisms,  76. 

-  sowing  cultures,  70. 

—  sterilization,  4. 

Methylene    blue-eosin    solution    (Chenzin- 
sky's),  210. 

-  (Romanowsky's),  210. 
Mexican  fever,  847. 

Miahle's  reaction,  262. 

Mice  as  experimental  animals,  156. 

-  handling  of,  162. 

-  coccidiosis  in,  764. 

-  epizootics  among  (B.  typhi  murium), 
444. 

-  favus  in,  692. 

-  Trypanosomes  in,  808. 


INDEX 


883 


Micrococcus  catarrhalis,  651. 
-   and  influenza,  504. 

—  fetidus,  578. 

—  gazogenes  alcalescens,  578. 

—  mammitis,  615. 

—  melitensis,  475. 

—  neoformans,  769. 

—  pelleteri,  665. 

—  salivarius  pyogenes,  617. 

-  tetragenus,  631. 

—  —  aureus,  632. 

—  —  concentricus,  632. 

ruber,  632. 

septicus,  632. 

subflavus,  632. 

—  —  variabilis,  632. 
Micrometer,  stage,  121. 

—  ocular,  122,  123. 
Micromonas  mesnili,  840. 
Micromyces  hofmanni,  669. 
Microscope,  106. 

—  care  of  the,  117. 

—  compound,  109. 

—  objectives,  107. 

—  stand,  106. 

Microscopical  appearance  of  micro-organ- 
isms (see  the  several  chapter  headings). 

—  image,  aplanatic,  110. 

—  objects,  measurement  of,  121. 
Microsporum  audouini,  688. 

—  —  var.  canis,  688. 

—  —  —   equinum,  689. 

-  felineum,  689. 

—  furfur,  669. 

—  minutissimum,  666. 
Microsporon  mansoni,  670. 
Microsporidia,  752. 
Microsporidium  apis,  753. 

—  bombycis,  752. 
Microtomes,  211. 
Miescheria,  756. 
Miescher's  tubes,  757. 
Milk  as  culture  medium,  35. 

-   -agar  for  gonococcus,  640. 

—  litmus,  57. 

—  acid  fast  bacilli  in,  338,  345. 

—  antitoxin  in,  266. 

—  B.  enteritidis  aertrycke  in,  438. 

—  collection  of  sterile,  35,  201. 

—  goats',  and  Mediterranean  fever,  475. 

—  tubercle  bacilli  in,  detection  of,  345. 
Miniopterus  schreibersii,  781. 

Miquel's  flask,  29,  852 

—  method  of  isolating  micro-organisms, 
76. 

—  —  examining  air,  866. 

—  peptone  solution,  33. 

—  serum  filter,  48. 

—  stain  for  spores,  147. 
Mistbazillus,  345. 
Molluscum  contagiosum,  766. 

of  birds,  842. 

Monas  pyophila,  827. 

Monkeys  as  experimental  animals.  157. 

-  care  of,  158. 

—  handling  of,  164. 

—  hsematozoon  of,  781. 


Monkeys,  spontaneous  plague  in,  460. 

—  tuberculosis  in,  294. 
Monocystis,  795. 

—  tenax,  796. 
Monobromonaphthaline,    refractive    index 

of,  119. 

Monovalent  serums  in  streptococcal  infec- 
tions, 606. 

Morel  and  Dulaus'  stain  (Actinomycosis), 
658. 

Moreschi's  typhoid  toxin,  378. 

Moro's  percutaneous  test  (Tuberculosis), 
328. 

Moser's  antistreptococcal  serum,  608. 

Mosny's  pneumococcal  serum,  586. 

Mosquitos,  examination  of,  780. 

-  and  equine  piroplasmosis,  792. 

-  and  malaria,  776. 

—  and  Mediterranean  fever,  476. 

—  and  oriental  sore,  802. 

-  and  plague,  462. 

—  and  sleeping  sickness,  820. 

-  and  yellow  fever,  841. 
Moulds,  parasitic,  675. 

—  for  paraffin  embedding,  214. 
Motility  of  bacteria,  152. 
Morax's  bacillus,  511. 
Mordants,  136. 

Mouth  sickness,  839. 

Much's  stain  (Tubercle  bacilli),  307. 

Mucor,  676. 

—  corymbifer,  677. 

—  mucedo,  676. 

—  racemosus,  677. 
Mucoracidce,  675. 
Mucormycosis,  677. 

Mucous  membranes,  inoculation  of,  171. 

Muffle  furnace,  17. 

Muhinyo,  475. 

Muir's  stain  for  flagella,  152. 

Mules,  Nagana  in,  811. 

Miiller's  method  of  isolating  the  typhoid 
bacillus,  406. 

Muscardine,  677. 

Musgrave  and  Clegg's  method  for  cultivat- 
ing amoebae,  750. 

Mycetoma,  662. 

—  parasites  of,  665. 
Mycotic  pityriasis,  670. 
Myxidium  danilewskyi,  756. 

—  lieberkuhni,  756. 
Myxobolus  biitschU,  756. 

—  cerebralis,  756. 

—  cyprini,  756. 

—  lintoni,  756. 
Myxococcidium  stegomyice,  754. 
Myxosporidia,  754. 

N.A.,  112. 

Naegeli's  medium,  39. 

—  method     for     isolation     of     micro- 
organisms, 76. 

Nseggerath    and    Staehelin's    method    for 

examining  blood  (Syphilis),  733. 
Nagana,  trypanosome  of,  811. 
Nahrstoff  Heyden,  317,  340. 
Nastikow's  violet,  139, 


3K2 


884 


INDEX 


Nastikow's  agar  medium  (Gonococcus),  639. 
Nattan-Larrier  and  Bergeron's  method  of 
examining  blood  (Syphilis),  733. 
-   (Tuberculosis),  342. 
Natural  immunity,  221. 
Needles,  glass,  for  sowing  cultures,  70. 

—  for  syringes,  168. 

-  (intra-peritoneal        inoculation), 
174. 

Negri  bodies,  841. 

Negro  lethargy,  816. 

Neisser's  stain  (diphtheria  bacillus),  252. 

—  (modified),  252. 

Neisser  and  Shiga's  dysentery  endo toxin, 
361. 

—  —  antityphoid  vaccine,  382. 
Nencki's  test  for  indol,  374. 
Neutral  red  as  an  indicator,  56. 

agar,  397. 

-  —   media,  411. 
Nicolle's  carbol-thionin,  138. 

-  carbol-gentian- violet,  138. 

-  capsule  stain,  148. 

-  decolourizing  solution  (Gram's  stain), 
143. 

-  iodine  solution,  209. 

-  mordant  (Gram's  stain),  143. 

-  stain  (simple)  for  sections,  217. 

-  (differential)  for  films,  208. 

-  sections,  210,  219. 

-  (Gonococcus),  637. 

-  culture  medium  (agar-gelatin),  43. 

-  (diphtheria  toxin),  260. 

-  (protozoa),  799. 

-  and    Duclaux's    method    of   cardiac 
puncture  (rabbits),  195. 

-  and  Morax's  stain  for  flagella,  151. 

-  and  Weil's  medium  for  leprosy,  353. 

-  Nikiforoff's  stain  (spirochsetes),  715. 
Nitrogen  in  growth  of  anaerobes,  89. 
Nitroso-indol  reaction,  494. 

N.N.N.  medium,  799. 

Nocard's  glycerin-potato   (Tubercle  bacil- 
lus), 320. 

—  trocar,  48. 

Nocard    and    Roux's     medium     (Pleuro- 

pneumonia  of  cattle),  837. 
Nocardia  actinomyces,  656. 

—  astero'ides,  662. 

-  farcinica,  667. 

-  forsteri,  662. 

-  hofmanni,  669. 

—  madurce,  662. 
Noeggerath's  culture  medium,  57. 
Noguchi's  medium  (Spirochaetes),  716. 

-  (Trepon,ema  pallidum),  737. 
Non-mannite-fermenting  dysentery  bacilli, 

357. 

Non -specificity  of  complement,  229. 
Normal  soda  solution,  31  footnote. 
Nosema  apis,  753. 

-  bombycis,  752. 

-  bryzo'ides,  754. 

-  ovoideum,  754. 

Novy  and  MacNeaFs  medium,  799. 
Numerical  aperture,  112. 
Nyctotherus  faba,  831. 


Objectives,  microscope,  107. 

—  apochromatic,  115. 
Ochroid  mycetoma,  665. 
Oculars,  compensating,  115. 
(Esophageal  catheterization,  181. 
Oididce,  701. 

Oidiomvcosis,  706. 
Oidium,  674. 

—  albicans,  702. 

—  lactis,  674. 

—  subtile  cutis,  674. 
Onychomycosis,  679,  686. 
Oospora  astero'ides,  662. 

—  bovis,  656. 

—  canina,  692. 

—  farcinica,  667. 

—  forsteri,  662. 

—  hofmanni,  669. 

—  lingualis,  706. 

—  madurce,  662. 
Oosporidce,  655. 

Ophthalmo-reaction  in  tuberculosis,  327. 
Opilasao,  822. 

Oppenheim  and  Sach's  stain  (T.  pallidum), 
729. 

Opsonic  index,  determination  of,  240. 

Opsonins,  223,  239. 

Optimum  temperature,  isolation  by  cultiva- 
tion at,  84. 

Oriental  sore,  802. 

Ornithodoros  moubata,  and  Tick  fever,  712. 

—  —   and  Spirochceta  marchouxi,  718. 

—  savignyi,  and  Tick  fever,  712. 
Orszag's  stain  for  spores,  147. 
Orth's  carmine,  218. 

—  alcohol  carmine,  218. 

—  picrocarmine,  218. 
Ose,  69. 

Osmic  acid,  fixation  of  films  with,  205. 

"  Osteomalacia  "  of  horses,  792. 

Ovoid  bacterium,  446. 

Oxen,  Nagana  in,  811. 

Oxycyanide  of  mercury,  sterilization  with, 

26. 
Oxygen,  action  of,  on  anaerobes,  87. 

—  tests  for,  92. 

Padlewsky's     method     of     isolating     the 

typhoid  bacillus,  411. 
Paget's  disease  of  the  nipple,  766. 
Pappenheim's  stain,  220. 
Paracolon  bacilli,  420. 
Para-dimethyl-amido-benzaldehyde  test  for 

indol,  374. 
Paraffin  for  sealing  tubes,  30. 

—  embedding,  212. 

—  moulds,  214. 

—  -xylol  mixture,  212. 
Paramcecium  coli,  830. 
Parasites  in  tumours,  766. 

-  of  Actinomycosis,  660. 

—  Mycetoma,  665. 
Paratyphoid  bacilli,  420. 

—  —   as  cause  of  epizootics,  160. 

—  A.  bacillus,  423. 

-  B.  bacillus,  431. 
Paratubercle  bacilli,  345. 


INDEX 


885 


Paris  vibrio,  491,  492. 

Park's  egg  medium  for  the  tubercle  bacillus, 

317. 
Park    and    Williams'    diphtheria    bacillus, 

257. 

-  toxin,  260. 

-  method  of  neutralization,  31. 
Passive  immunity,  222. 

Pasteur  flasks,  29. 

-  pipettes,  67. 
Pasteur's  hot  air  sterilizer,  5. 

-  method  of  cultivating  anaerobes,  92. 

-  examining  air,  862,  865. 

-  tube  for  cultivating  anaerobes,  93. 

-  septicaemia,  86,  561. 

-  synthetic  medium,  38. 

-  vaccines  for  anthrax,  528. 

-  wine  medium,  38. 
Pasteur-Chamberland  filter,  14. 
Pasteur,  Joubert  and  Chamberland's  tube 

for  cultivating  anaerobes,  93. 
Pasteurella  group  of  bacilli,  446. 

—   compared  with  plague,   447, 
464. 

-  infections,  vaccination  against,  459. 
Pasteurella  bovis,  455. 

—  canis,  457. 

—  caprce,  456. 

—  cuniculi,  453. 

-  equi,  456. 

-  gallince,  447. 

—  ovis,  456. 

—  suis,  454. 

Pasteurellosis,  spontaneous,  among  labora- 
tory animals,  160. 

—  and  plague  in  animals,  447. 
Pasteurization,  12,  45. 

Pastor's    method    of    cultivating    tubercle 

bacilli  from  sputum,  340. 
Pastilles  of  diphtheria  antitoxin,  269. 
Pebrine,  752. 

Pediculus  capitis  and  Spirochsetosis,  711. 
and  Typhus,  848. 

—  vestimenti    (corporis)    and    Spirochse- 
tosis, 711. 

-   and  Typhus,  848. 
Pelikan  Tusche,  83. 
Penetrating  power  of  lenses,  113. 
Penicillum,  700. 

-  crustaceum,  700. 

-  glaucum,  700. 

—  minimum,  700. 

-  pictor,  698. 

Peptone  (see  also  Agar  and  gelatin  culture 
media). 

-  solution  (Gordon's)  for  carbohydrate 
reactions,  600. 

(Koch's),  33. 

-  (Martin's),  32. 
-l.ccf-broth,  30. 

-  -broth  (Martin's),  33. 

—  -gelatin      medium      (Metchnikoff's), 
33. 

—  -yeast  extract,  37. 

Perchloride    solution    (Schaudinn's)    as    a 
fixative,  749. 

—  —   for  sterilization,  26. 


Pere's    method    of   isolating    the    typhoid 

bacillus,  402. 
Perisporacidce,  694. 
Perlsucht,  294  footnote. 
Percutaneous  reaction,  328. 
Pernicious  anaemia  of  horses,  456. 

—  malaria,  780. 
Pertussis,  bacillus  of.  511. 
Petri  dish,  55. 

Petri's  method  of  examining  air,  865. 

—  cholera  toxin,  494. 

Petzval's  condition  for  flatness  of  image, 

115. 
Pfeiffer's  cholera  serum,  499. 

-  influenza  bacillus,  504. 

-  phenomenon,  222,  227,  499. 

-  warm  stage,  135. 

Pfeiffer  and  Kolle's  antityphoid  vaccine, 
381. 

Phagocytosis,  222. 

Pharyngeal  exudates,  collection  of,  197. 

Pheasants,  epizootics  among  (Past,  gal- 
lince), 447. 

Phenol-phthalei'n  as  indicator,  31. 

Phisalix's  collodion  sacs,  176. 

—  egg  medium  for  the  tubercle  bacillus, 
317. 

-  vaccines  for  distemper,  458. 
Phlebotomus  and  oriental  sore,  802. 
Pian,  736. 

Pianese's  fixative  for  coccidia,  761. 

Picrocarmine,  Orth's,  218. 

Piedra,  670. 

Pigeon  crammers'  disease,  696. 

Pigeons  and  plague,  461. 

Pigeons,  epizootics  among  (Past,  gallince), 

447. 
Pigs,  Sarcosporidia  in,  756. 

—  Tuberculosis  in,  295. 

—  and  plague,  461. 

—  contagious  pneumonia  of,  454. 
Pigs'  stomach  broth,  32. 
Pigments  of  B.  pyocyaneus,  279. 
Pincushion  distortion,  109. 

Pinta,  698. 
Pipettes,  bulb,  22. 

—  Levaditi's,  234. 

—  Pasteur,  67. 

—  Roux,  92. 

—  feeding  experiments  with,  181. 
Piroplasma,  786. 

—  bigeminum,  787. 

—  canis,  791. 

-  equi,  792. 

—  mutans,  793. 

—  ovis,  791. 

—  parvum,  793  footnote. 

—  pitheci,  793. 

von  Pirquet's  cuti-reaction  in  Tuberculosis, 

327. 

Pitfield's  flagellum  stain,  152. 
Pithion    and    Roux's    stain    for    tubercle 

bacilli,  309. 

Pityria-sis  versicolor,  66! ». 
alba,  670. 

—  —   flava,  670. 
'nigra,  670. 


INDEX 


Placenta  as  culture  medium,  54. 

—  -agar      for      Meningococcus      (Kut- 
scher's),  647. 

—  -broth  for  tubercle  bacilli,  318. 
Plagiomonas  ir regular  is ,  827. 

—  urinaria,  827. 
Plague,  bacillus,  460. 

-   differentiation    from    pasteurella 
bacilli,  447. 

—  spontaneous,  in  rabbits  (post  mortem 
appearances),  454. 

—  and  swine  plague,  454. 

—  infection  of  rats  by  feeding,  464. 
Plasmodium,  770. 

—  danilewskyi,  782. 

—  malarice,  770,  780. 
—  prcecox,  770,  780. 

—  relictum,  782. 

—  vivax,  770,  780. 

—  ziemanni.  782. 
Plaster  plates  for  filtering,  14. 
Plates  Bombicci's,  101. 

—  Kitasato's,  101. 

—  Zinsser's,  102. 
Platinum  wires,  69. 

Plato's  stain  (Gonococcus),  637. 
Pleural  fluid,  collection  of,  198. 

—  —  as  culture  medium,  52. 
Pleuro-pneumonia    contagiosa,     virus     of, 

836. 

Pneumobacillus  of  Friedlander,  415. 
Pneumococcus,  580,  601. 

—  medium  for  (blood  broth),  34. 

—  and  influenza,  504. 
Pneumonia,  gonococcal,  634. 

—  spontaneous,  in  animals,  160. 
Pneumonic  plague,  460,  462. 
Pneumo-enteritis  of  horses,  456. 
Pockenkrankheit,  756. 
Polychrome  blue,  Unna's,  139. 

—  discomyces  of  Vallee,  669. 
Polyvalent  antistreptococcal  serum,  608. 
Pommeliere,  294  footnote. 

Ponos,  800. 

Porous  porcelain  filters,  14. 

Porges'  serum  diagnosis  of  syphilis,  741. 

Porges  and  Meyer's  diagnosis  of  syphilis, 

740. 

Portal  vein,  inoculation  into,  177. 
Porospora  gigantea,  796. 
Post  mortem  examinations,  184. 

—  —  infections,  Colon  bacillus  in,  393. 
Post  partum  sepsis  in  cows,  Gaertner  bacil- 
lus in,  442. 

Potain's  aspirator,  52. 
Potassium  pyrogallate,  89. 
Potato  bacillus,  isolation  of,  84. 

—  medium,  37,  55. 

—  —  synthetic,  56. 

—  —  —  (Remy  and  Sugg's),  372. 

—  tube,  55. 

Pouchet  and  Bonjean's  method  of  isolating 

the  typhoid  bacillus,  402. 
Pravaz's  syringe,  166. 
Precipitins,  227. 
"  Premier  vaccin  "  of  Pasteur  (Anthrax), 

528. 


Preventive  strength  and  antitoxic  strength 

(diphtheria  antitoxin),  268. 
Primary  agglutinins  (see  Agglutinins). 
Proca  and  Vasilescu's  stain  (T.  pallidum), 

729. 

Prophylactic  serums,  223. 
Prophylaxis  of  tetanus,  546. 
Proteosoma,  770. 

—  of  Labbe,  782. 

Proteus  hominis  capsulatus,  571. 
Protozoic  dermatitis,  706. 
Prurigo  decalvans,  688. 
Pseudo-acid-fast  bacilli,  346. 

—  -actinomycosis,  665. 

—  -alopecia,  692. 

—  -diphtheria  bacillus,  273. 

—  -gaertner  bacilli,  444. 

—  -gonorrhoea,  634. 

influenza  bacilli  (Pfeiffer's),  510. 

—  -miliary  tuberculoses,  346. 

—  -mucin,  639  footnote. 
Pseudonavicella,  795. 

Pseudo- paratyphoid  A.  bacillus,  430. 

— •   -tuberculosis  in  experimental  animals, 

160. 
bacillus  of,  160,  474. 

—  -tuberculoses,  347. 
Psittacosis,  445. 
Psorospermiasis,  706. 
Psorospermosis  follicularis,  766. 
Puerperal  septicaemia,  592. 

-  serum  treatment  of,  607,  608. 
Pulex  (Xenopsyllus)  cheopis  and  plague,  461. 

—  -irritans  and  plague,  461. 

-  murinus  (X.  cheopis),  461. 

—  pallidus  (X.  cheopis),  461. 

-  philippinensis  (X.  cheopis),  461. 
Pulmonary  anthrax,  517. 

—  exudates,  collection  of,  198. 

—  mycosis,  677. 

-  rhizomucormycosis,  678. 
Pump,  d'Alvergniat's,  90. 

—  mercury,  90. 

—  vacuum,  90. 

—  water,  90. 
Puncture  of  spleen,  198. 
Pus,  collection  of,  197. 

-  detection  of  tubercle  bacilli  in,  342 
Pysemia,  617. 

Pyocyanine,  279. 
Pyocyanolysin,  280. 
Pyogenic  staphylococci,  617. 
Pyrosoma,  786  footnote. 

—  bigeminum,  787. 

Qualitative  examination  of  water,  856. 
Quartan  malarial  fever,  780. 
Quarter  ill,  552. 

-  vaccines  (Arloing's),  556. 

—  —   (Leclainche  and  Vallee's),  557, 
558. 
Quotidian  malarial  fever,  780. 

Rabbits  as  experimental  animals,  156. 

—  bleeding  of,  194. 

—  epizootics  among  (Gaertner  bacillus), 
442. 


INDEX 


887 


Rabbits,  epizootics  among  (Past,  gallinca), 
44o* 

—  —  —   ( —  cuniculi),  453. 

-  (Plague  baciUus),  460. 

—  handling  of,  161. 

—  inoculation  into  the  biliary  passages 
of,  177. 

—  in tra-cranial  inoculation  of,  180. 

—  intra-spinal  181. 

-  intra- venous  172. 

-  cesophageal  catheterization  of,  181. 
Rabbit  pasteurellosis,  453. 

—  septicaemia,  bacillus  of,  453. 
Rabbits,  spontaneous  trypanosomiasis  of, 

808. 

—  —   tuberculosis  of,  295. 
Rabies,  virus  of,  841. 

—  simulated  in  dogs  by  infection  with 
B.  pyocyaneus,  276. 

Rsebiger's  stain  for  capsules,  148. 
Rag-pickers'  disease,  561. 
Rainey's  tubes,  757. 
Raisin-gelatin,  41. 
Ramond's  agar,  397. 
Ramsden  eyepiece,  122  footnote. 
Ranvier's  hollow-ground  slide,  134. 

—  lamp,  118. 

—  rat  bit,  163. 

-  warm  stage  for  the  microscope,  135. 
Rats  as  experimental  animals,  156. 

—  handling  of,  162,  163. 

-  epizootics  among  (B.  gaertner),  442. 

-  (B.  typhi  murium),  444. 
(Leprosy),  348. 

—   (Plague),  460. 

—  Post  mortem  appearances, 
474. 

-  spontaneous  methsemoglobinsemia  in, 
442. 

Rat  louse  and  Trypanosoma  lewisi,  805. 

-  viruses,  445. 
Raulin's  culture  medium,  38. 
Ravaut's  stain  (T.  pallidum),  728. 
Ravaut  and  Ponselle's  method  of  examining 

the  blood  (Syphilis),  733. 
Razors,  microtome,  211. 
Re-activated  bacteriolytic  serum,  228. 
Rectal  injections,  182. 
Red  mycetoma,  665. 
Red  water  of  cattle,  787. 
Reed  sacs,  176. 

Refractive  index  (cedar  wood  oil,  etc.),  119. 
Reitmann's  stain  (T.  pallidum),  729. 
Relapsing  fever,  711. 
Remy's     "  differential    gelatin "     for    the 

typhoid  bacillus,  404. 
Remy  and  Sugg's  stain  for  tiagella,  150. 
-  synthetic  medium,  375. 

-  potato  medium,  372. 
Resolving  power,  112. 

Resolution  with  dark-ground  illumination, 

123. 
Reservoirs  of  Sleeping  sickness,  820,  821. 

-  Trypanosoma  brucei,  811. 
Rheumatism,  acute,  592. 

—  bacillus  (Achalme's),  569,  570. 

—  gonorrhoeal,  634. 


Rhinosporidium  kinealyi,  759. 
Rhipicephalus  appendiculatus  and  Theileria 
parva,  793. 

—  bursa  and  Piroplasma  ovis,  791. 

—  decoloratus   and   Spirochceta   theileri, 
719. 

—  evertsi  and  Piroplasma  equi,  792. 

—  sanguineus    and    Piroplasma    canis, 
791. 

—  simus  and  Theileria  parva,  793. 
Rhizomucor  parasiticus,  678. 
Rhizopoda,  745. 

Rhizopus  cohni,  678. 

—  equini,  678. 

—  niger,  678. 

—  nigricans,  678. 
Rhodesian  fever  of  cattle,  793. 
Ribbert's  stain  (Pneumococcus),  584. 
Rice  milk  culture  medium,  56. 
Rinderpest,  virus  of,  839. 

Ring  parasite  of  malaria,  774. 
Ringworm,  679. 

—  in  cats,  687. 

—  dogs,  687,  688. 

—  fowls,  687. 

—  horses,  687. 
Rocking  microtome,  211. 
Rocky  Mountains  fever,  802. 

Rodet's  method  of  isolating  the  typhoid 

bacillus,  402. 

Rodents,  Trypanosomes  in,  808. 
Rohrbeck's  temperature  regulator,  59. 
Roll  tube,  Esmarch's,  81. 
Romanowsky  blood  stain,  210. 
Roosen-Runge's   method   of  isolating   the 

typhoid  bacillus,  392. 
Rosenthal's  antidysentery  serum,  362. 
Rosenbach's    Streptococcus   pyogenes,    593, 

596. 

Rosenbach's  stain  (T.  cruzi),  822. 
Rosette  parasite  of  malaria,  775. 
Ross'  method  of  blood  examination 

(malaria),  772. 

de  Rossi's  stain  for  Hagella,  157. 
Rost's  medium  for  leprosy,  352. 
Roth's  method  of  isolating  the  typhoid 

bacillus,  409. 

Rothe's  serum-broth  medium,  274. 
Rougeole,  283  footnote. 
Rouget  du  pore,  283  footnote. 
Roux  bottle,  78. 
Roux's  blue,  140. 

—  carbol-crystal-violet,  138. 

-  gelatin,  40. 

-  incubator,  65. 

—  pipette    for    cultivating    anaerobes, 
92,  99. 

-  potato  tube,  55. 

-  syringe,  167. 

-  temperature  regulator,  60. 

—  tube  for  cultivating  anaerobes,  100, 
101. 

—   isolating  anaerobes,  103. 

-  standardization   of  diphtheria   anti- 
toxin, 268. 

Roux,  Metchnikoff  and  Salimbeni's  cholera 
toxin,  495. 


888 


INDEX 


Roux  and  Martin's  diphtheria  toxin,  258. 

Roux  and  Nocard's  method  of  preparing 
serum,  48. 
—   reed  sacs,  176. 

Rowland's  plague  toxin,  467. 
-  serum,  472. 

Royal   Commission    on    Tuberculosis,    290 
et  seq. 

Ruediger's    ascitic    agar    (Pneumococcus), 
585. 

Ruffer's  method  tor  demonstrating  para- 
sites in  cancer,  768. 

Saathoff  s  differential  stain  for  sections,  220. 
Sabouraud's  glucose-agar  (Sporotricha),  673. 

-  glycerin-agar   (Achorion   schaenleini), 
692. 

-  proof  agar  (Tricophyta),  681. 

-  agar  (Tricophyta),  681. 
Sacs,  collodion,  174. 

—  reed,  176. 
Saccharomyces,  701  footnote. 

—  albicans,  702. 

-  angince,  705. 

-  ellipsoideus,  706. 

-  granulatus,  706. 

-  guttulatus,  706. 

-  lithogenes,  706. 

-  membranogenes,  706. 

-  neoformans,  707. 

-  roseus,  706. 

-  tumefaciens,  704. 

—  and  Cancer,  707. 
Saccharomycetidce,  701. 
Saccharomycosis,  706. 
Sahli's  borax  blue,  681. 
Salkowski's  reaction  for  indol,  374. 
Salmonella  group  of  bacilli,  422,  431. 

—  and  plague,  473. 
Salomonsen's  agar,  44. 
Sanarelli's  typhoid  toxin,  377. 
Sand  as  a  triturating  agent,  170. 
Sand  flies  and  Oriental  sore,  802. 
Sanfelice's  antianthrax  serum,  532. 
Sarcocele  in  glanders,  481. 
Sarcocystine,  758. 

Sarcocystis  blanchardi,  758. 

—  immitis,  756,  758. 

—  miescheriana,  758. 

—  muris,  756,  758. 

—  tenella,  756,  758. 
Sarcosporidia,  756. 
Sarcosporosis  in  man,  756. 

—  pigs,  757. 

—  rats,  758. 

—  sheep,  756. 

Saturation  of  agglutinins,  436. 
Savage's  neutral  red  media,  411. 

Scarlet  fever  and  streptococci,  592,  596,  607. 
Schaudinn's    classification    of    Trypanoso- 
midce,  804. 

-  fixative,  749. 

-  stain  (T.  pallidum),  730. 
Schizotrypanum  cruzi,  822. 

Schmitz,  Turro  and  Blell's  cholera  toxin, 
495. 

—  —  —   —   vaccine,  497. 


Schottmiiller's  classification  of  Streptococci, 

599. 
Schueder's  method  of  isolating  the  typhoid 

bacillus,  406. 

Schiiffner's  dots,  773,  780. 
Schwein  Rothlauf,  283  footnote. 
Schweinepest,  438,  454  footnote. 
Schweineseuche,  454. 
Sclavo's  stain  for  flagella,  153. 

—  antianthrax  serum,  532. 
Schlerothrix  kochi,  289,  661. 
Seborrhoea  oleosa,  692. 

Secondary  agglutinins  (see  Co-agglutinins). 
"Second  vaccin"  of  Pasteur  (Anthrax),  528. 
Sections,  histological,  211. 

—  fixation  of,  215. 

—  staining  of  (simple),  216. 

—  —   (differential),  217. 

—  —  —   gram      negative      organisms, 
220. 

-  (triple),  219. 
Section  lifter,  215. 
Sensibilisatrice,  228. 

Sensitized  vaccines  (Besredka's)  in  Dysen- 
tery, 362. 

-  Enteric  fever,  383. 
Plague,  470. 

-  Streptococcal  infections,  605. 
Septicaemia,  Pasteur's,  86,  561,  563. 

-  spontaneous,  in  animals,  159. 
Serous  exudates,  examination  of  (Tubercle 

bacilli),  342. 
Serum,  ascitic  fluid,  52. 

—  blood,  36. 

—  coagulation  of,  51. 

—  collection  of,  45,  196. 

—  glycerin,  53. 

—  Loeffler's,  52. 

—  pleural  fluid,  52. 

—  -agar,  53. 

-  (Bordet  and  Gengou),  511. 

-  (Tochtermann's),  53. 

-  -broth,  34. 
Serums,  agglutinating,  225. 

—  antitoxic,  224. 

—  bactericidal,  227. 

—  haemolytic,  230. 

—  prophylactic,  223. 

-  therapeutic,  223. 

Serum   diagnosis   (see  the  several  chapter 
headings),  225. 

—  therapy    (see    the    several    chapter 
headings),  225. 

Sewage,  bacteriological  examination  of,  861. 
Shanghai  vibrio,  492. 
Sharpening  of  razors,  211. 
Sheep  pasteurellosis,  456. 

-  piroplasmosis,  791. 

-  and  plague,  461. 

—  sarcosporidiosis,  756. 

-  spirochsetosis,  719. 

—  Tuberculosis  in,  295. 

-  -pox,  Virus  of,  839. 
—   cell,  840. 

Shiga's  antidysentery  serum,  362. 

-  vaccine,  362. 

—  antiplague  vaccine,  470. 


INDEX 


889 


Shiga-Kriise  dysentery  bacillus,  357. 
Siberian  fever  of  horses  (Anthrax),  578. 
Silk,  preparation  of  sterile,  165. 
Simonelli  and  Bandi's  stain  (T.  pallidum), 

729. 

Simple  continued  fever  in  India,  423. 
-   staining  solutions,  137. 

-   of  films,  205. 
Simulium  (Genus),  699. 
Sine  condition  for  aplanatism,  110. 
Sivori's  trocar,  48,  49. 
Skin,     removal     of,     for     bacteriological 

examination,  191. 
Slatineano's  influenza  toxin,  508. 
Slides,  cleaning  of  microscope,  130. 
Sleeping  Sickness,  816. 
Smear  preparations,  203. 
Smegma  bacillus,  346. 
Solid  substances,  inoculation  of,  169. 
Sobernheim's  antianthrax  serum,  532. 
Soft  sore  and  syphilis,  576. 
Solid  media,  39. 
Sowing  of  cultures,  67. 
Souma,  814. 
Soudakewitch's  method  for  demonstrating 

parasites  in  cancers,  767. 
Spatula,  215. 

—  (Drigalski's),  407. 

Specific  agglutinins  (see  Agglutinins). 
Spengler's  method  for  cultivating  tubercle 
bacilli  from  sputum,  341. 

—  stain  (Tubercle  bacilli),  309. 
Spherical  aberration,  110. 
Spirillum  nigrum,  577. 
Spirilla,  356. 

Spirochceta  (Genus),  804. 

—  anserina,  717. 

—  balanitidis,  734. 

—  buccalis,  735. 

—  carteri,  712. 

—  dentium,  735. 

—  duttani,  712. 

—  gallince,  718. 

—  kochi,  712. 

—  Icewenthali,  736. 
— -  marchouxi,  718. 

—  media,  735. 

—  microgirata,  736. 

—  neveuxi,  718. 

—  nicolei,  718. 

—  novyi,  712. 

—  oberm.eyeri,  712. 

-  pallida,  720. 

—  pertenuis,  736. 

—  plicatilis,  735. 

—  recurrentis,  712. 

-  refringens,  734. 

-  rossii,  712. 

—  theileri,  719. 

—  vincenti,  735. 

Spirochaetes  of  malignant  ulcers,  735. 
Spirochsetosis,  711. 
Spironema,  720. 
Splenectomy,  199. 
Splenic  apoplexy,  517. 

—  fever,  517. 

—  puncture,  198. 


Spontaneous     diseases     of     experimental 

animals,  159. 
Sporadic  dysentery,  356. 
Spores,  145. 

—  resistance  to  heat,  7. 

—  staining  of,  146. 

Spore-bearing  organisms,  isolation  of,  84. 
Sporo-agglutination  (Sporotrichosis),  674. 
Sporotrichosis,  672. 

Sporotrichum  beurmanni,  672. 

—  dori,  672. 

—  schenki,  672. 

Sprengler's  method  of  examining  sputum 

(Tubercle  bacilli),  339. 
Spronck's    medium    for    diphtheria    toxin, 

257. 

—  yeast  extract,  37. 
Sputum,  collection  of,  191. 

-  homogenization  of,  339. 
Squirrels,  Trypanosomes  in,  808. 
Stab  cultures,  sowing  of,  72. 

—  —   characters  of,  74. 
Stage,  Koch's  drying,  141. 
Stains  for  micro-organisms,  136. 

—  aniline  fuchsin,  483. 

—  carbol-fuchsin-methylene-blue  (Quey- 
rat),  514. 

—  cresoidin,  253. 
Staining  of  blood  films,  207. 

-  capsules,  147. 

—  film  preparations,  140,  205. 

—  flagella,  148. 

—  living  organisms,  140. 

—  sections,  216. 

—  spores,  146. 
Stalactites  in  plague,  465,  473. 
Standardization  of  antitoxin  (diphtheria), 

267. 

—  —  (tetanus),  545. 

—  antityphoid  vaccine,  381. 
Staphylococci  pyogenetes,  617. 
Slaphylococcus  cutis  communis,  693. 

—  parvulus,  578. 

—  pyogenes  albus,  617,  620. 

-  aureus,  617,  619. 
citreus,  617,  620. 

Staphylolysin,  623. 

Starch  jelly,  56. 

Stassano's  apparatus  for  collecting  blood, 

196. 

Steam,  sterilization  in,  7,  9. 
Steamers,  8. 
Stegomyia  fasciata,  Nosema  in,  754. 

-  and  yellow  fever,  841. 
Stephens'  stain  for  flagella,  153. 
Sterilizers,  hot  air,  5. 
Sterilization,  definition  of,  3. 

—  by  antiseptics,  26. 

—  discontinuous  heating,  8,  12. 

—  dry  heat,  4. 

—  filtration,  14. 

—  moist  heat,  7. 

Stern's  stain  (T.  pallidum),  728. 
Sterygmatocystis,  699. 

-  nidulans,  665,  699. 

-  nigra,  699. 
Stomoxys  and  Surra,  814. 


890 


INDEX 


Stomoxys  calcitrans  and  anterior  poliomye- 
litis, 846. 

Stools,  collection  of,  for  bacteriological 
examination,  202. 

Strangles,  611. 

Straus'  stain  for  flagella,  148. 

—  "  sign  "  in  glanders,  487. 

—  syringe,  166. 

Straus  and  Wurtz's  method  for  examina- 
tion of  air,  866. 
Straw  infusion,  37. 

Steinschneider's  agar  for  Gonococcus,  640. 
Streptococci  hominis,  593. 

—  animalium,  611. 

—  classification  of  (Andrewes  and  Hor- 
der),  601. 

—  varieties  of,  593. 
Streptococcus  of  Bonome,  610. 

—  anginosus,  601. 

—  brews,  593,  596. 

—  conglomeratus,  596. 

—  equi,  611. 

—  equinus,  601. 

-  erysipelatos,  593,  596,  599. 

—  fcecalis,  601. 

—  longus,  593,  596. 

—  meningitidis,  610. 

-  mammitis  bovis,  613. 

—  mitior,  599. 

-  mitis,  601. 

-  mucosus,  599. 

—  pyogenes,  593,  599,  601. 

—  salivarius,  601. 

—  tennis,  593,  596. 

—  viridans,  599. 
Streptocolysin,  603. 
Streptothrix  (see  Discomyces),  655. 

-  pleom orphic,  in  leprosy,  351. 

—  actinomyces,  656. 

—  astero'ides,  662. 

—  caprcB,  668. 

—  farcinica,  667. 

—  fdrsteri,  662. 

—  freeri,  664. 

—  israeli,  661. 

—  leproides,  348. 

—  madurce,  662. 

—  rosenbachi,  662. 

—  spitzi,  661. 
Streptothricine,  660. 

"  Strict "  anaerobes,  87. 
Strong's  cholera  vaccine,  498. 

-  dysentery  bacillus,  360. 
Stroke  cultures,  method  of  sowing,  71. 

—  —  isolation  by,  81. 
Sub-cutaneous  inoculation,  171. 
Surra,  814. 

Susceptibility  of  animals  to  experimental 

inoculation,  156. 
Swans,  pasteurellosis  in,  452. 
Swine  erysipelas,  bacillus  of,  283. 

—  fever,  virus  of,  843. 

—  pasteurellosis,  454. 

—  plague,  bacillus  of,  454. 

—  tuberculosis,  295. 

—  typhoid,  454  footnote. 
Sycosis,  686. 


Symptomatic  anthrax  (see  Quarter  ill),  552. 
Synthetic  media,  38. 

—  medium  (Remy  and  Sugg's),  372. 
Syphilis,  720. 

—  bacillus  of  Lustgarten,  306  footnote, 
346. 

Syringes  for  inoculation,  166. 
Syringospora  robini,  702. 

Tabanus  and  Surra,  814. 
Tannin  solution  (Nicolle's),  210,  217. 
Tarbagan  and  Plague,  462. 
Tarozzi's  method  of  cultivating  anaerobes, 
98. 

—  —   isolating  anaerobes,  102. 
"  Tarse  favique,"  691. 

Tavel,  bacillus  of,  306  footnote. 
Tavel's  antistreptococcus  serum,  609. 

-  stain  (Tubercle  bacilli),  306. 
"  Teigne  tondante  "  of  Gmby,  688. 

rebelle,"  688. 

Telosporidia,  760. 

Temperature  of  cattle,  182  footnote. 

—  regulators,  59. 

Terni  and  Bandi's  Plague  vaccine,  469. 
Tertian  fever,  780. 
Tests  for  indol,  374. 

—  oxygen,  92. 
Tetanolysin,  544. 
Tetanus  bacillus,  536. 

—  antitoxin,  545. 

—  prophylaxis,  546. 
Texas  fever,  787. 
Theileria  parva,  793. 
Therapeutic  serums,  223. 
Thesing's  stain  for  spores,  147. 
Thrush,  702. 

Thymol  as  an  antiseptic,  26. 

Thymus  broth,  34. 

Tick  fever,  712. 

Ticks  and  Piroplasmata,  786  et  seq. 

—  and  kala  azar,  800. 

—  and  Rocky  Mountains  fever,  802. 
Timothee  bazillus,  345. 

Tinea  barbce,  679. 

—  circinata,  679. 

—  —  dysidrosiforme,  687. 

—  cruris,  688. 

-  imbricata,  687,  700. 

—  kerion,  686. 

—  marginata,  688. 

—  rosea,  670. 

—  sycosis,  679. 

—  tonsurans,  679. 

—  versicolor,  669. 

Tissues,  bacteriological  examination  of,  203. 

—  as  culture  media  (Tubercle  bacilli), 
318. 

Titre  of  agglutination,  measurement  of,  388. 
Tochtermann's  agar,  317. 

—  serum -agar,  53. 
Tokelau,  700. 

Toluene  method  of  embedding,  215. 
Toluol  as  an  antiseptic,  27. 
Tonsil,  puncture  of,  197. 
Tortoises,  tuberculosis  in,  297. 
Toxicity  and  virulence,  257  footnote. 


INDEX 


891 


Toxin  (see  the  several  chapter  headings). 

—  unit  of,  260. 
Trachea,  inoculation  into,  179. 
Tray  for  sloping  culture  media,  52. 
Trenkmann's  stain  for  flagella,  151. 
Treponema  (Genus),  804. 

-  pallidum,  720. 

—  pallidulum,  736. 

Tretrop's  vacuum  apparatus  for  cultivating 

anaerobes, 
Trichomonas,  356. 

-  batracorum,  827. 

-  cavice,  827. 

—  intestinalis,  825. 

—  vaginalis,  825. 
Trichosporosis,  670. 
Trichosporum,  670. 

-  beigeli,  671. 

-  foxi,  671. 

-  giganteum,  671. 

—  krusi.  671. 

-  ovale,  671. 

-  ovo'ides,  671. 
Tricophyton,  679. 

-  acuminatum,  684. 

-  a^teroides,  685. 

-  caninum,  687. 

-  concentricum.  687. 

-  crateriforme,  682. 

-  ectothrix,  686. 

-  megaspores,  682. 
—    inicroides,  682. 

-  endothrix,  682. 

—  endo-ectothrix,  682,  685. 

-  equinum,  687. 

—  faviforme,  687. 

—  felineum,  687. 

—  fragile  mycelium,  684. 

-  gypseum,  682,  685. 

—  mansoni,  687. 

-  megnini,  687. 

-  megalosporum  endothrix,  682. 

-  mentagrophytes,  685. 

-  microsporum,  688. 

-  niveum,  682,  687. 

-  ptctor,  698. 

-  pyogenes,  686. 

-  radians,  687. 

-  resistant  mycelium,  683. 

-  sabouraudi,  684. 

-  sulphureum,  685. 

-  tonsurans,  682. 

—  inolaceum,  685. 

Triple  staining  of  sections,  219. 

Tristeza,  787. 

Tse-tse  fly  disease,  811. 

-  flies  and  Nagana,  811. 

-  and  Sleeping  Sickness,  820. 
Trocar,  Nocard's,  48. 

-  Sivori's,  48. 
Tropical  malarial  fever,  780. 

-  piroplasmosis  of  cattle,  793. 
Trout,  disease  of,  756. 
Trypanoplasma  (Genus),  804. 
Trypanosoma  (Genus),  804. 

-  abramis,  825. 

—  avium,  823. 


Trypanosoma  brucei,  811. 

-  cazalboui,  814. 

—  congolense,  813. 

—  cruzi,  822. 

-  danilewskyi;  825. 

—  dimorphon,  813. 

—  equinum,  814. 

—  equiperdum,  809. 

—  evansi,  814. 

—  gambiense,  816. 

—  lewisi,  805. 

—  pecaudi,  814. 

—  ro/cB,  825. 

—  re-mo**,  825. 

-  rhodesiense,  820. 

—  rotatorium,  824. 

-  rougeti,  809. 

—  sanguinis,  824. 

—  solece,  825. 

—  soudanense,  813. 

—  theileri,  816. 

—  ugandense,  816. 
Trypanosomata,  802. 
Trypanosome  fever,  816. 

—  of  Dourine,  809. 

—  —   Galziekte,  816. 
Mai  de  Caderas,  814. 

—  —   Nagana,  811. 

-  Surra,  814. 
Trypanosomes  of  Sleeping  Sickness,  816. 

—  in  birds,  823. 

-  cold-blooded  vertebrata,  824. 

-  fish,  824. 
frogs,  824. 

Tube,    Buchner's,    for    cultivating    anae- 
robes, 95. 

-  Esmarch's,   for  isolating  anaerobes, 
103. 

—  Turro's,  for  cultivating  anaerobes,  95. 

—  Vignal's,  for  isolating  anaerobes,  103. 
Tubercle  bacillus,  289. 

-  avian,  291. 

—  bovine,  289. 

-  human,  289. 

—  ichthic,  292. 

-  differentiation  of  types  by  cultiva- 
tion, 320. 

Tuberculin,  324. 

—  test,  325. 
in  man,  326. 

Tuberculosis,  Royal  Commission  on,  290  et 

seq. 
Tumours,   removal  of,   for  bacteriological 

examination,  198. 

—  and  Coccidia,  766. 
Turkeys,  Pasteurellosis  in,  447. 

—  and  plague,  461. 

Turro's  tube  for  cultivating  anaerobes,  95. 

-  method  of  isolating  anaerobes,  101. 

-  gelatin  medium  for  Gonococcu.s,  638. 
Twort's  medium  (Leprosy  bacillus),  352. 
Tyndallization,  45. 

Typhoid  bacillus,  366. 

—  carriers,  367. 
Typholysin,  379. 

Typhus  diagnosticum,  Ficker's,  388. 
Typhus  fever,  virus  of,  847. 


892 


INDEX 


Udder-broth  (Micrococcus  neoformans),  769. 
Ultra-microscope,  123. 
Ultra-microscopic  viruses,  835. 
Undulant  fever,  475. 
Undulina  ranarum,  824. 
Unit  of  antitoxin,  268. 

— •   measurement   for   microscopical   ob- 
jects, 122. 

—  toxin,  260. 

Unna's  polychrome  blue,  139. 
Ureter,  inoculation  into,  178. 
Urethral  inflammations,  634. 
Urinary  baciUus  of  Clado,  393. 
Urine  as  culture  medium,  36. 
(Gonococcus),  638. 

—  collection  of  sterile,  201. 
Uschinsky's  medium  for  diphtheria  toxin,  39. 

Vaccination  (see  the  several  chapter  head- 
ings). 
Vaccines,  Wright's,  605;  623. 

—  Besredka's    "sensitized,"    362,    383, 
470,  605. 

Vacuum  incubators,  104. 

-  pump,  90. 

Vaillard  and  Dopter's  antidysentery  serum, 

362. 
Vallee's     method     of    immunizing     cattle 

against  Tuberculosis,  332. 
Vallet's   method  of  isolating  the  typhoid 

bacillus,  406. 
Varieties  of  the  dysentery  bacillus.  357. 

-  streptococci,  593. 

-  the  tubercle  bacillus,  290. 
Variola  ovina,  839. 

—  vaccinia,  840. 

Vasilescu's      homogeneous      cultures      of 

tubercle  baciUi,  336. 
Veal  broth,  32. 
Vegetable  media,  37,  55. 
VeUlon's  method  of  isolating  aerobes,  82. 
—   anaerobes,  103. 

—  Streptococcus  tenuis,  596. 
Verruga  peruana.  bacillus  of,  346. 
Versailles  vibrio,  490. 

Vesuvin,  aqueous  solution  of,  208,  218. 
Viability  of  micro-organisms  (see  the  several 

chapter  headings). 
Vibrio  cholerce  asiaticce,  488. 
Vibrio  of  Deneke,  503. 

—  Finkler-Prior,  502. 
Vibrio  metchnikowi,  503. 
Vibrion  avicide,  503. 

—  septique,  561. 

Vignal's  tube  for  isolating  anaerobes,  103. 

—  warm  stage,  135. 

Villemin  type  of  experimental  tuberculosis, 

298,  300. 
Vincent's  angina,  574,  575. 

—  method    of    isolating    the    typhoid 
bacillus,  85,  402. 

—   examining  stools  for  amcebse.  748. 

—  stain  for  blood  films,  207. 

-   ( Bacillus  fusiformis),  576. 
Vincent  and  Beliefs  reaction,  650. 


Violet,  Nastikow's,  139. 
Virulence     of    micro-organisms     (see    the 
several  chapter  headings). 

—  and  toxicity,  257  footnote. 
Vitality  of  micro-organisms  (see  the  several 

chapter  headings). 

—  phagocyted  bacteria,  222. 

Wahl's  stain  (Gonococcus),  637. 
Warm  stage  for  microscope.  135. 
Wassermann's  antityphoid  vaccination,  382. 

—  reaction  in  Syphilis,  737. 

Water,  bacteriological  examination  of.  851. 

—  nitration  of,  15. 

—  baths,  12. 

—  pump,  90. 

Weeks  and  Morax's  bacillus,  510. 

Weigert's  solution,  143. 
'• —  stain  for  sections,  216. 

Weil's  culture  medium  (Leprosy),  353. 

Werbitzki's     medium     for     isolating     the 
typhoid  bacillus,  410. 

Werner's  typhoid  toxin,  378. 

Wertheim's  agar  (Gonococcus),  638. 

West  African  relapsing  fever,  713. 

Weyl-Legal's  test  for  indol,  374. 

White  mycetoma,  665. 

Whooping  cough,  bacillus  of,  511. 

Wild  and  Rinderseuche,  455. 

Wildbolz's  agar  (Gonococcus),  639. 

Windelbandt's    method    of    isolating    the 
typhoid  bacillus,  412. 

Wine  as  a  culture  medium,  38. 

Woolsorter's  disease,  517. 

Wright's  capsule  for  collecting  blood,  192. 

Wright's  vaccines,  605,  623. 

Wright  and  Leishman's  antityphoid  vac- 
cination, 381. 

Xenopsyllus  cheopis,  461. 
Xerosis  bacillus,  245  footnote. 
Xylol  method  of  embedding,  212. 

Y  bacillus  (dysentery),  360. 

Yaws,  736. 

Yeasts,  701. 

Yeast  extract  medium  (Spronck's),  37. 

Yellow  fever,  754,  841. 

—  and  Bacillus  icteroides,  445. 
Yersin's  gelatin-broth  (Plague),  465. 

—  plague  serum,  471. 

Yersin  type  of  experimental  tuberculosis,. 
301. 

Zabolotny's  stain  (T.  pallidum),  729. 
Zeidler's  method  of  isolating  the  typhoid 

bacillus  from  blood,  391. 
Zeihl's  carbol-fuchsin,  138. 
Zeihl-Neelsen's  stain  (Tubercle  bacilli),  307, 

310. 

Zinsser's  method  of  isolating  anaerobes,  102. 
Zoogleic  pseudo-tuberculoses,  347. 
Zygospores,  675. 
Zymonema,  701  footnote. 
-   gikhristi,  706. 


GLASGOW  :    PRINTED   AT  THE   UNIVERSITY  PRESS   BY  ROBERT   MACLEHOSE   AND  CO.  LTD. 


